University of the Pacific Scholarly Commons

University of the Pacific Theses and Dissertations Graduate School

1972

The chemical analysis of Velella Lata float

Ralph Lee Gainey University of the Pacific

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Recommended Citation Gainey, Ralph Lee. (1972). The chemical analysis of Velella Lata float. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/1772

This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. THE CHEMICJ\L ANALYSIS OF 'VELELLA LATA li'LOAT

A Thesis Presented to l the Faculty of the Graduate School University of the Pacific

l In Partial Fulfillment j of the Requirements for the Degree I ! Master of Science

by Ralph Lee Gainey November 1972 ACKNOWLEDGEIIJEN'r

The author wishes to acknowledge:

the courage of Dr. Otis Shao, who opened the door; the faith of Dr. Emerson Cobb, who kept it open; and the dedication of Dr. Paul Gross, who gen­ erously provided'space in his labor- atory and patiently demonstr- I ated its effective use in the direction l of this work. 1 DEDICNI'ION

T'hin thesis is dedicated to:

i l Dr. riLMfred R:iJnpler'

of Hannover, Germany, whose encouragement and technical guidance are appredated

more than he knows;

a11d to:

Carol

my wife, who knows . l I TABIE OF CONTENTS

CHAPTER PAGE

1. Introduction • • . • • . . . • I I 0 I I I I o I 1 A. General Considerations of Nolecular Architecture • . . • . • • . • • 1 B. Components of Connective Tissue 3 C. Investigation of Chitin 5 D.· Velella lata . 7 2. Experimental Pr•ocedure 11 A. Determination of Moisture Content 13 Summary ...... I • 0 • 15 B. Deterrn:lnation of Inorgamc Content 16

Sl..liiDl8..cy • • • • • • • I I 0 • • 20 C. Determination of Ether Soluble Material 21' Stl!T!rrlar'".Y • • • • • • • • • • • • • • 22 D. Determination of Carbohydrate Content s~~ ...... E. Determ:ix1ation of Protein Content Swrmary , . .

3. Brief Summary of Results

Ll. Discussion of Results 50 A. Moisture Content 50 B. Inorgamc Content 53 C. Lipid Content 58 D. Carbohydrate Content 60 E. Protein Content 64

5. Conclusion 71 References 76 Appendix . 82 I 1 I l IJ.ST OF FIGURES 1 FIGURE PAGE ~ l. Velella lata . • • • • . • • • • . • Sa

1 2. Relationship of Cell Layers to Float Sa j 3. Orthogonal Projection of Velell~Float 8b I 4. Detail of Horizontal and Vertical MarginS Sb

1 5. The Time Dependence of the Water Uptake of Dried KOH-Treated Float Material ...... • • • 15a 1 6. Detennination of Optimum Hydrolysis Conditions 2Sa 1 7. Colorimetric Detennination of Carbohydrate 29a

1 S. 'Ihin Layer Chromatography • I I I I I I I 32a I 9 . Hypothetical MacromJlecule Containing Six Lysine- i L:inl{ed Octasaccharides . . • . . • ...... 99

10. HypotheU.cal Construction of Velella lata Float Based on a Topologically Continuous Concentrlc Membrane Model • . .100 LIST OF TABlES

TABLE PAGE

1. Amino Acid Results ...... • . • 39 2. Protein Found in Am1no Acid Analysis 40 3. Calculation of Protein and Carbohydrate Ratio 40

4. Lysine Residue in Standards and KOH-Treated M.'l.tei'ial 42 5. Ratios of Lysine to Other Amino Acids 43 6. Mole Ratio of Lysine to Carbohydrate • 43 7. Mole Ratio of Protein to Carbohydrate 411 8. Systematic Identification of Velella lata 97 9 . Phyla of the An:ilnal Kingdom ...... • 98 l

\ I LIST OF JJ1FRARED SPEC'l'HA l j SPF.£TRUM PAGE l 1. N-acetylglucosamine (a)*. 83 -l (b) 84 2. Glucosamine hydrochloride (a) 85 J (b) 86 3. Shrimp Shell (a) 87 (b) 88 1 4. Velella Float Material I .\b'N KOH, 8 days at 25°C) (a) 89 j (b) 90 1 . 5. Velella Float Material (lN ROi1, 24 br at l00°C) (a) 91 (b) 92 6. Crystal:Li.!1e Haterial from Acid Hydrolysate of' Vele._lla Float Materie.l (a) 93 (b) 94 7. Velella Float Material (waghed and dried) (a) 95 (b) 96

*(a) 500-1200 em-1 (b) 1200-3500 cm-l "Thus, the cellular fll!lction of the multi­ cellular organism cannot be understood fully without complete lmowledge of the structure

and fUnction of the (extracellular) matrix."

l INTRODUCTION -~ 1 :1 A. General Considerations of Molecular Architecture Our universe exhibits a unity which extends to all observable

1 phenomena, and allows us to confidently propose the structure of the

1 hYdrogen molecule as the basis for a code to communicate with in-

j telligent beings anywhere within it. rl'his unity is expressed l l nowhere more dramatically than in the world of living things, where ' all of the physical laws are obeyed and a continuous speetrum of molec-

j ular evolution exists. A con~sr:Lson of proteins r~~cm different an:i.Inals J l shows amazing similarities :Ln the amino acid sequences of molecules engaged in similar functions (i.e. the oxygen carrying molecules, pituitary hormones, and various insulins)~ 1 The more closely r·elated

the two animals, the more similar the proteins are. The distinction

between two species is primarily based on macroscopically observable

differences in shape and pigmentation, which we expect to be caused

by molecular vaPiation at the site. Differences in behavior are usually

considered an adaptation to the environment and in lieu of shape and

pigment differences are seldom sufficient for the discrimination into

a new species or subspecies. Examination of biological structure

reveals a heirarchy of levels of organization from the small to the

large and the simple to the complex. In cotton, for example, the 2

organization is pentafid: . glucose molecule, fibril, microfibril,

macrofibr.il, and cotton hair?

Everything should be ultimately explaimble in terms of the

components of which it is composed. In the animal kingdom well over

ninety-five percent of all recognized species are invertebrates, a

distinction based on connective tissue, though most of our efforts

have been directed towards vertebrates for reasons of convenience

and closeness to man. Investigation of the various chemicals which

make up plants and animals has been diverse, allowing us to get a

general pict-ure of what molecules to expect at each stage of the

evolutl.omry progression,· but for no animal 1s the information complete,

and for invertebrates the information is particularly sketchy. It

would be convenient to have certain animals and plants completely

lmovm cherrd.cally, so that biological molecules subsequently discovered

could be compared to these standarc1c;. Velerla lata rnav be a candidate . --- " for such a standard, having a number of unique advantages. It occurs

in the evolutionaJ:y chain at that point where a true multicellular

animal begins, and it is the simplest animal from which genetically

determined inte1~al structural material is conveniently isolated m1d

purified.

In plants the cormective tj_ssue is considered to be almost en-

tirely carbohydrate polymer and in vertebrates the connective tissue

is considered to be almost entirely protein (amino acid polymer),

while many :invertebrates have aflnost an even mixture of protein- j carbohydrate in an unknown relationship. Velella presents an oppor- -1 I l 3

tunity to examine closely the nature of the protein and its relation- I ship to the carbohydrate. l The carbohydrate of various invertebrate connective tissue matrices ~ l has been extensively studied and the structure and linkages reasonably· J well defined. Some questions remain regarding the length of the

1.I I smallest oligosaccaride and the manner in which these are built up

'\ into higher levels of organization. Inorganic Inaterial (primarily in the form of calcium), lipld in small amounts, and moisture are also 1 .j present in the invertebrate matrix. While caldum is generally con- 1 th~t "I sidered to be in the form of the carbonate, the possibility it is j not all in this form awaits investigation, and the recent report that

silicon is essential in the formation of chick connective tissue is

interesting. 5 Lipids m'e not a major component in connective tissues

studied to date, and this is true of Velella also. This does not _j 1 preclude, however, the possibillty that lipids may play some role in

the formatlon of membrane-like sheets which al'e observed in the lam-

inations of var'ious mollusk-·arthropod matrices. Velella offers an

opportunity to examine invertebrate connective tissue in an intense

way to gain an understanding of the morphology and developmental

dynamics which hopefully would be extensible to the vertebrate matrix.

'rhe present picture of biologkal activity indicates that all

events originate in the genetic code of phosphate-sugar polymers in

the celJ. nucleus. The code is apparently only for proteins, to the

exclusion of lipids, car'bohydrates and other non-protein molecules. The most obvious visible expression of this genetic code is shape 4

and pigmentati.on, and the presence of non-protein molecules

1 in the matrix leaves a gap in our understanding. The mediation of l proteins (especially as enzymes) in species-specific events is observed

in the biosynthesis of matri.x precursors and is expected to determine I in some fashion the final products. Self-assembly (aggregation, J ! polymerization, crystallization) is a corrmon phenomenon of cellulose~ 2 l chitili~,lB and collagerr~' 4 \he matrix materials of widest distribution. The self-assembly of both intracellular and extracellular "organelles" '1 is receiving increasing attention. Only two or three "forms" of

l chitin are organized into a great variety of shapes, and one is tempted 1 to account for this anomaly in terms of an attached protein moiety. J ] Can the extracellular self-assembly of specific macromolecules account ' entirely for the specificity of the matrix? To what extent is the

adjacent ,cell layer a factor? Cell surface prote:tns are involved

in the aggregation of di.ssociated cells29 •47 a,':ld specH'ic carbohydrates are identified with the irregu.lariUes exhibited by neoplastic cells~ 9 7 3 Glycoproteins are corrmonly isolated from rr.atrix materials~ • %urely these facts are related in some way. The golgi apparatus apparently

functions to synthesize or organize these protein-carbohydrate com­

plexes3B,56 and this suggests a pathway between DNA and final shape.

If this sequence is generally accurate it should be possj_ble to define

in detail the reactions jnvolved and demonstrate the presence of

the necessary intermediates and enzymes. ye1ella appears to be an

animal sufficiently simple to facilitate this , yet complete enough

to illustrate the generality of the scheme; a solid matrix is present I without problems of isolation and purificat1on. 'l'he related "protein" ! ! 5 l I may be polymerized and branched in an intricate way (e.g. by disulfide

j and £-lysine crosslinks) so that what we conceive as an individual l amino acid polymer becomes a monomer at a higher level of organization. l It is thus of interest to examine in detail the invertebrate matrix 1 j proteins, of "'hich resilin, arthropodin and sclerotin have so far been 1 1\ isolated and studied. The protein from Velella lata float, which j may be called "velatin", can now be added for comparison. 'I'he sequenc- I ing of these structural proteins may reveal the phylogenetic progression 1 we have learned to expect . j j C. The Investigation of Chitin

l The histor~ and occurrence of chitin have been recently reviewed~9

I The identification of the carbohydrate moiety as the ~. HIJ linked I 2" 40 polymer of N"·acetylgJ.uc:osamJne seems wen established/' Not all

-, of the peaks in the i:C' spectrum have been identified, 35 but the absorption at 891:!: 7 cm-l is characteristic of' the beta linkage.35 The purest f'onn was found in the internal skeleton of the squid. 19 Chitin ~1as

found to give three distinct x-ray diffraction patterns which were

designated alpha, beta and gamma. Beta and garruna were convertible

into alpha.18 ' 19 The beta form was interpreted to bdicate the mono-

hydrate, with the water molecule bound between the C-3 of one N-

acetylglucosaminyl unit and an hydroxymethyl group in an adjacent 10 chain.

Elemental analyses have not settled the degree of acetylation.

However, Hunt found no free amino groups using dinitrophenylation.19

'I'he aggregation of chitin wUh various peptides and amino acids in I I 6 j solution was studied and the coJI[Jlexes formed were found to be sensitive

I to ion:i.c strength and.pH, completely dissociating above pH 9. 17 I The gener'a1 occurrence of a protein moiety tightly associated with ~ chitin has been found and studied in attempts to identify the nature

of the linlmge and the amino acid(s) involved. 19 Asparagine and 18 histid.i.ne were found still associated after 60 hr in lN NaOH at. 100°C,

and ch:i.ttn dtssolved :i.n saturated Uthium thiocyanate conta:i.ned protein

when repredpttated by the addition of acetone.13 Several studies

indicated that the carbohydrate-protein linkages tdentified so far

invariably involve the anomeric carbon of the carbohydrate. 19 4 4 ~he oligosaccharide length has been studied in depth~• • ° Column chromatography of acid hydrolysates separated the first tleV("Y1 homologous 2 oligosaccharides. Polarized ir ev:tdence supports x-·I.'<,y DO\•Jder d:i.agrmm:;

\·Ihich gaye a- repeat cli.st.anee of J~ l 1\. for· the axial di:cect.ion > col:'reB- I.J2 pondj_ng .to etght N--acetylglucosaminyl units.

X-ray powder d:tagrams were interpreted to indicate that the cha:tne

are oriented so that adjacent cha:tns are :i.n the same direction, but

that alternate pairs of chains are oriented in the opposite direction.6

This intcerpretation was supported by polarized ir work which tlhowed

a lack of free OH and NH g;roups and no C=O· · · HO bond:LYJg. 7 A structure

occurred in the powder diagrams from a variety of alpha and beta

0 42 chitins at repeat distances of 31 A, corresponding to six monomers.

This repeat survived boiling in benzene for 11 0 hrs . Electron m:tcro·-

graphs seemed to show a regular hexagonal array of carbohydrate sur- 42 0 rounded by protein. Gamma ch:i.tin showed the repeat of 41 A and was l 7 l j interpreted to indicate a slightly different spatial array, and

li removal of the protein gave the usual alpha chitin pattern after ' 42 spontaneous aggregation.

There are similarities between collagen and chitin. Invertebrate 16 collagens high (10-15%) in carbohydrates and hydroxylysine linkages50

have been found. A glycoprotein is reported to be always linked to 21,27 coll

·contains a large amount of lysine. 43,49 A macromolecule identifled

as glucosylgalactosy1hydroxylysine is reported "widely distributed 24 throughout the animal kingdom as the carbohydrate moiety of collagen."

The present study supplements many of these findings in an interesting way.

D. Vele11a lata

The full systematic identification of Velella 1_ata is given in

the Appendix (Tables 8,9). It is a coelenterate (Order hydroida) 8

which occurs in the Pacific Ocean as the pelagic, free~ floating medusa

(figure 1).23 It is generally considered to be a colony of polyps 20 (cup-like fonns). It appears as a small (3-5 em) pig;nented jellyfish

and has the common names "purple sailor" and "by-the-wind sailor''.

Velella is rarely found in the intertidal region but onshore storms

often sweep the surface-dwelling animals onto the beaches of N. California

and Oregon, and the dried floats are sometimes found in rows several

inches thick along the beach for miles. Masses of Velella have been . 23 reported at sea to extend for 260 km. References for the complete

distribution were not found, but world-wide ten species are known. 23

~~ta is a closely related animal also having a. disc-shaped float,

but without the vertical "sail" component. The orientation of the

sa.il is 40° to the wind (j_dentical to PhysaHa.... the Portugese man-

of-war) and is the sole means of transportation. The survival value

of this stn1cture may be inferr•3d from the discovery of Velella floats 8 in rocks dated 450 million years old. ' 12 The occurrence of solid

structural material in the lower animals is rare and the presence

of the invertebrate analogue of bone in a "jellyfish" is strar.ge

indeed.. Invertebrates commonly exhibit what is generally termed

an "exoskeleton", while Velella ).ata has a "float" (figure 2,3) which

is completely internal. 'The formation has been viewed as an exo-

skeleton formed internally by a folding over and inward of the external

cell layer. The float material as found on the beach is a light tan I or dull white, turning yellow :i.n the sun. The yellowing is associated j with the protein moiety and when it is removed the carbohydrate is a 1 Sa

end view j side view

1 I

J Figure 1. Velell~ lata l l vertical margin (narrow grove)

external cell layer(s) -""""' 1111-1--.. "sail" of flcra t

me soglea ·-' ·float horizontal margin (wide, flat groove) -septa \.

tentacles.- gastrozooid

cross section

l'igure 2, Relationship of Cell Layers to Float I Bb li I l l J end side Il j 1 I life size 1 i top

Figure 3. Orthogonal Pro·jection of Yelella Float

margin~

horizontal

:float ----­ (shown delaminated for illustration ) vertical cross section

Figure 1}, Detail of Horizontal and Vertical Margins 9

clear dull white. The untreated float gives a negative Molisch test

for carbohydrate. The float from formalin-preserved specimens separates

easily from the fleshy tissue much like a foot from a shoe, with no

apparent connection to the adjacent cell layers. Careful examination

did not reveal any connection to the fleshy tissue, nor any associated

membrane systems . The float material seems to be laid down L'1 layers

in a cyclical manrier. The edges of the float are extended by an in-

crease in the diameter of the muscle-like peripheral tissue, which 20 1-Iymsn states is contractile. Examination of floats separated from

formalin-treated specimens revealed that the float is constructed 1 by the laying down of flat sheets of material, each slightly larger

than the last, resulting i.n a concentrically symmetrical structure 1 ·I ' wH.h concentric lines where the overlap between layers oc;cur·s (figure l\). ~'he form,_ql:in-treateO. float material readily "peels" apart J.ike an on:'. on, _j 1 revealing the 1dentical, thoug_l-J smaller, float beneath it. Tlle layers 1 ! of'the vertical component are continuous witll those of the horizontal component so that the synthet1c activ1ty of the entire cell layer

seems coord1nated. The horizontal component d1ffers from the vertical

in; that 1t contai.ns monolayered septa wll1ch result from the f;:t.at

surface of the horizontal marg:inalgr>oove (figure 4). The vertical

edge is sharp, and apparently the float i.s bilaminar along the ver-

tical ridge, although these layers could not be separated (figure Li) •

It appears that the entire adjacent cell layer• i.s engaged in synthesidng

float material subuni.ts and these then spontaneously or1ent (self-

assemble?) themselves into the flat sheets which adhere smoothly ' --1 l 10

to the previous layer (figure 10). Growth apparently involves a

cycle of mitosis-synthesis, which may be circadian. Such light­

related phenomena have been reported in the biosynthesis of chitin 30 . in an anthropod skeleton. The ubiquitious distribution of a purple

pigment in invertebrates suggests the possibility of an energy-trapping

system such as occurs associated with the green chlorophyll pigment

of plants. Photosynthesis is sometimes defined as the direct utili­

zation of light energy for the biosynthesis of carbohydrate polymers. 51

Since chitin is a carbohydrate, might not such a system be suspected? 1 Enzymes in a suggested carbohydrate biosynthetic pathway 9 are in­

directly light sensitive (by their ATP requirements) and aequorin,

a calcium-specific pr·otein which occurs in other jellyfish, biolUIJLtnesces 48 . in the presence of free calcium - also a component of the watrix.

_.,' The float is central to all of these questions and its chemical analysis 1 1 fonns the basis of the pr•esent study. I i I

l EXPERJJViENTAL I l Preface

Weights were taken on a Mettler anf.llytical balance (type Hl5)

provided with silica gel desiccant in the weighing chamber. Crucibles

and glassware .used in weight determinations were pre-rinsed in a H 2so4 mixture. All glassware was cleaned by washing with detergent, rinsing

with distilled water and with an acetone/methanol mixture and dried

at ll0°C in the oven for at least ten minutes. Weights were ta~en

after the crucibles and glassware had cooled to constant weight in

1 the wej_ghing chamber. Infrared spectra were taken on a Perkin-Elmer 1 337 spectrophotomet.:~r, on KBr pellets at a concentration of l-2mg/

100 D'.g F.Br. Amino acid analyses were carried out on a Bio-cal 201

P.mino Acid Analy;;ei', using the single colum technique. 'l'lc was ca-rried

out on glass plates coated with silica gel G and GF (2:1) by developing

in one of two solvent systems:

System #1; CHC1 , MeOH, EtOH (90/5/5) 3 System #2; n-propanol, ethyl acetate, NH40H (25%), H2o (80/10/10/30) Spots were visualized by spraying wHh H (10% in MeOH) and charring 2so4 in an oven at 125-150°C. Ele.mental analyses were done by Microanaly-

tisches Laboratorium Beller, Gottingen, Germany. Melting points were

determined on a Thomas-Hoover "Unimelt" apparatus and m'e uncorrected.

In ~ac~1o means less than 0.1 mm Hg unless otherwise specified. "Wig- L-·Bug" is made by Crescent Dental !llanufacturing Co. , Chicago, Illinois.

The words "float" and "float material" are used interchangeably and

refer to the entire float without distinctj.on between the vertical and 12

! horizontal. components. The material described is that which is easily I separable from the intact animal ru1d survives washing with water. j I -I " 1

1 I 1 l I I l CHAPI'ER 2

l EXPEIID1ENTAL PROCEDURE PART A

Determination of Moisture Content

Whole Float

Over:. caq_2. ApproY.imately 2 g of diced float material were carefully washed in' tap water and dilute HCl (J.N) to free it of sand and extraneous

material. After being rinsed in dist:i.lled water until neutral, the

float material was allowed to dry in air until the weight was constant.

In a crucible the float material (1. 8696 g) was then placed in a con-

stant temperature desiccator over CaC12 at 60°C at less than 0.1 mm Hg. 'Iwo days later the material was allowed to cool to room tempePature

and was quickly weighed. The loss of weight ( 0 .1927 g) was 10.30%

of the weight of the float material befol'e drying.

~r P2%: Whole float material (0.9184 g) which had been diced, washed and dried in air as above, was placed in a test tube (constant

·weight), and dried over P o at 65°C and less than 0.1 mm Hg. After 2 5 67 hrs the float mater·ial was weighed at room temperature ( 0. 7851 g) .

The loss of weight (0.1333 g) was 14.5% of the weight of the float

nuterial before drying.

-1 14

~H Treated Float Ma!erial ll Over P 2~. Appr9ximately 0 .1 g of float material which had been freed I of protein by the Rudall method (lN KOH at 100°C for 24 hr), washed ! in distilled water until neutral, and dried in the lab atmosphere to l constant weight, was added to one vial. After weighing, three vials ~ wlth float material were dried over P o at 65°C and less than 0.1 mm r 2 5 Hg. After 75 hr heating, the float material was allowed to cool to 1 I room temperature in the drying pistol. After the material was removed, I it w:as quickly weighed. The weight loss of each vial, calculated as

a percentage of the weight of the float material was: #1 - 14 .14%; I #2- ---14.10%; #3- 15.3%.

!'ei'l9EJ?t1on of Moiiiture by KOH Treated PloB:t ~1aterial. Vials #2 and #3

were cove.red with alum:i.rn.m1 foil with small holes and kept in the lab

atmosphere. 'I'he vials were weighed periodically: ':rime-----·- in air Resorbed water (! of original loss) Vial #2 ViaUJ_ 4hr 40.5% 38.8% 10 hr 70.5% 74.1% 79 hr 78.5% 81.5%

'l'he two vials were then redried over P o in vacuo at 65°C as before 2 5 for 46 hr. After cooling to room temperature, the weights were

the same as on the previous drying. Again the two vials were allowed

to stand in the lab atmosphere as before and the weights periodically 15

I Resorbed Y.!_ater (% of original loss) l Vial #2 Vial #3 j 24 hr 75.8% 77.0% j 49 hr 83.3% 85.0% 82 hr 82.0% 83.8% 32 days 106.0% 104.0%

A graph of the resorption data was prepared (figure 5), which

indicated non-linear resorption.

Sumnar,y.

Loss on drying of whole float material

OVer CaC1 , 48 hr, 60°C in vacuo 2 OVer P o , 67 h~, 65°C in vacuo 2 5

I,oss_9]1 dr;Jir~ of KOH treated float material

J-4.1%

---j 14.1%

15.3% 15a

1 1 tB:n=rn+++ ,• 90 l (dried over P o 2 5 85 at 65°G ·in y_~) 80 - 11 1 -+-' -i 75 1 70

65 I I ~ \ -~rT- _I 60 ' ' l 55 l J ' ~ j I ___j

'I\ -t---t---t-1 I + 30 'ffi' I -~ '' ' ti H 25 4- - j . --L ' ' 20

..l __j_ --i L-t=t- -, ' j ' 15 _L t . ' -+ -+- •:tt ' _ij -~ 10 th I --1---L -:·- - + =H .++~tt l-- - I c-r- 'n- 8=· ·i [j;~$ JrjL[--- ' 5 -~f# .. L _u_l 10 20 30 40 50 60 70 80 90 100 % Oi' Lost Moisture Resorbed

Figure 5 THE TIME DEPENDENCY OF THE WATER UPTAKE OF DRIED KOH-'.rREA1'ED FLOAT MATERIAL EXPERIMENTAL PROCEDURE PARr B

Detennination of Inorganic Content

Materi.al Extracted From Whole Float Material With Water. Whole float

material which was c:ollected from the beach was washed with vigorous

shaking in distilled water only. The water extract was suction fil­

tered and evaporated in a Nickel crucible (heating gently over a bunsen

burner) to give 0.2146 g of residue. After ashing over a Meker burner

the residue was weighed again ( 0.1483 g; 22.,_Q% of the dry residue).

Clean crab shell which had been ground to a powder and treated

in a manner similar to the float material gave '72. 4%.

Whole Float Materi.al c--i I l ~ Sand Content. Float material was washed in distilled water, followed I j by decantation of' the suspended float material to separate out sand.

This process was repeated for a prolonged period, interspersed with II manual separation of any wood or other extraneous material. Very

thorough washing did not remove sand which was found to be trapped

within the float structure. The f'loat material was air dried on

aluminum foil. The weighed dry float m."lteria.l was placed in a. constant

weight crucible, a.shed to whiteness and the ash residue was determined.

Tbe results of several determinations, expressed as a percentage of' the

weight of the washed float material, were: 2. 8%, 3. O%, }..'7%. If' -l sand was trapped hi.gher percentages up to 111% could be found. ' 17

Float material,

extensively washed in distilled water, was cleaned and selected to be

free of visible sand grains. It was· shaken in a separatory funnel with

distilled water, and sand was removed through the stopcock. After l j blending with distilled water, in a Waring blender, the procedure· l H was repeated. Finally, the material was air dried and placed in a 'I platinwn crucible (0.8532 g). The crucible with float material was l put into a muffle furnace at ll0°C for 15 minutes, after which the l temperature was increased to 450°C for 2 hr. After allowing to cool

1 to room temperature, the ash weighed 0.0071 g (0.83% of the weight 1 of the washed and dried float material). l The crucible and ash were returned to the muffle furnace for l

l hr at 550°C and cooled to room temperature. The residue ( 0. 0045 g),

expressed as a percentage of the weight of the washed and dried float material, was ---0.528%. The residue was transferred wii;h cone BN0 (20 rnl), to a platinum 3 crucible. The solution was heated in the crucible to dryness, allowed

to cool to constant weight, (0.0196 g). Cone HF (10 ml) was added

and the residue was heated to dryness on an asbestos pad over a bunsen burner flame (natural gas), allowed to cool to constant weight and weighed.

The procedure was repeated twice with 5 rnl HF until the weight remained

constant (0. 0096 g). The weight of' the residue, expressed as a per-

cent age of the original starting material, was 1.13%. 18

Wet Ashi.!)£

Acid Digestlon. (1.8195 g) of the float material (free of visible

sand and other extraneous matter, dried in air) was treated with cone

HCl0 (10 ml). and cone HN0 (10 ml) and heated until the solution 4 3 was clear. A small amount of gray material rerr.ained suspended. After

removal of the acid by heating, the wei~~t of the residue was determined.

The residue, expressed as a percentage of the washed and dried float

mater:i.al, was £:]%, ( 2. 8% after 12 hr in air) .

Ashing to Wh:i.teness. The residue from (a) was quantitatively trans­

ferred to a platinum crucible, with cone HN0 , and heated to dryness 3 with cone HN0 and cone HClo (2-3 ml) several times, until the res:idue 3 4 was white. The residue, expressed as a percentage of tJ-,e weig.'1t of

t:l1e""'- wcoji.~dt:.\.0 -·'-'- .'~.!-and '"'"'v....L~:..-'.. .~d --'-'f'bai- '"' fn''te,...l'•• =..- a'..L.) •.v~ct,.;:) ,~~ -'-'''2 9"'

Co12c !lJ!'.]'reatmc;ni~: Th'2cWhite residue from (b) was further treated

by adCLi.ng cone Hfl (2-3 ml), heating to dryness and cooling to constant

weight. This treatment was repeated until the weights became constant (3X).

~'he residue was 2. 611% of the weight of' the washed and dr•ied float

material. Recalculation considering 14% moisture gave 3.1%. The.

platinum cruc:i.ble did not change weight during the analysis.

KOH •rreated F'loat Material

Mild KOH Treatment. F'loat material (washed, selected to be free of

visible sand material) was treated with lN KOH (l00°C for 24 hr),

rinsed in distilled water to neutrality, rinsed in acetone (3X), and I I ! l 19 1 I l allowed to dry in air on aluminum foil. 'l'his float material (approxi­ J mately 0. 2 g :· each) was weighed into three crucibles which were

placed in an electric furnace (about 400°C) until the ash was white

and the weight constant . 'l'he weight of the ash was determined by

difference. 'l'he results, expressed as a percentage of the weight

of the pre-ashed float material, were: 1.811%, 3.60.% and ).21%. 'l'he

residues were dissolved in hot cone HCl and transferred with washing

(:3<"0 to flasks to which armnonium oxalate was added. After several

hours, flask A had a barely perceptible precipltate, flask B a copious

precipitate and flask C, a moderate precipitate.

Intensive KOH Treatment. Float material which had been kept in 6N

KOH for 3 days at 100°C, washed with distilled water until neutral, ~ J rinsed with a.cetone (3X), and allowed to dry in air• on aluminum foil, l J 1-1as placed equally in three crucibles and ashed over a Meker burner i. to constant weig.ht. 'l'he weight of the residue was determined by

difference and found to be: 3.43%, 3.34%, and 7.67% (red ash).

Desiccated KOH Treated Material. A porcelain cruDible was brought

to constant weight. Float material, approximately 0.1 g, which had

been carefully washed, was treated with JN KOH (100°C for 24 hr),

rinsed with distilled water until neutral, rinsed in acetone (3X), and dried over P o at 65°C and less than 0.1 rim Hg, for 24 hr, was 2 5 placed in the crucible and was quickly weighed ( 0.1031 g). Now it

was ashed to constant weight in an electric furnace between 350-450°0 -l (several hr) . 'l'he weight of the residue at room temperature was 4 . 35% . I I I 20

Su:rrunaty CJ._f Inorganic Determination (Unless otherwise indicated, all percentages are of the air-·dry float material)

ful..~ Extract 69.0%

Whole Float Material (12!;y_ Ash)

Washed; visible sand present 2.8%, 3.0%, 3.7% 1 ~ Washed, blended and sorted; free of I visible sand I i 450°C.ash 0.8% 550°C ash 0.5% .I After HF treatment 1.1% 1 \\lhole Float ]Vlaterial (Wet_ Ash) l Cone HN0 /HCl0 2.7% 3 4 Reweigh after 12 hr 2.8% After treatmsnt with cone HN0 2.9% 3 _fi4fte~:~ t:P.eatment_ vli th. cone. HF 2.6% SubtmcUng 1~% moistm"e factor 3.1%

KOH 'rreated Float Material

lN KOH (211 hr at 100°C) (Oxalate positive for calcium) 1.8%, 3.6%, 3.2% 6N KOH ( 3 days at 100° C) 3.3% 3.4% (red ash) 7.7% Desiccated KOH treated float material 4 .li%

I -~ l j I 21 l l EXPERIMENTAL PROCEDURE PARI' C

1 Determination of Ether Soluble Material j First Determination. Whole float material to be extracted was cleaned ~ as before and dried in air to constant weight. Approxim3.tely 2 g was 1 weighed and was carefully transferred to the ether pre-washed cel1ulose I cup of a soxhlet into the extraction apparatus with pure diethyl I ether (approximately 75 ml). After 24 hr, the extracted float material was allowed to dry in air on aluminum foil. The extr•acted float l material was weighed and the loss of weight was 0.14% of the pre- l j extraction weight of the float material. Recalculation after sub-- ~ 1 tracting a. factor of J)l. 5% for moisture content gave a value of 0 .17%. l -'j ;

tion weight of the float material. After the extraction, the cup

and floa:i; material were placed in a desiccator over P at 65°C p 5 and less than 0.1 mm Hg. After three hr the apparatus was allowed

to cool to room temperature and the float naterial was removed and was

quickly weighed. The loss of weight expressed as a percentage of the

pre-extraction weight was 13.7%. The loss of weight due to ether

extr•action expressed as a percentage of the desiccated float material

was .Q_,_l7%. The ether solution was left for evaporation to constant weight and the remaintng light brown syrup was weighed. The weight

of the extracted material was detenuined by difference. The loss of 22

weight expressed as a percentage of the pre--extraction weight of the float material was 0. 23%. The loss of weight expressed as a per­ centage of the weight .of the desiccated float material was 0.27%.

1st Determination

Loss in weight of air-dried float material during ether extraction Recalculated after subtracting a 14.5% moisture content l factor from the weight of the air-dried sa'llple 0 .17% 2nd Determination

II Loss in weight of air-dried float ~aterial ] dm•ir,g ether extraction 0 .15% J ~I Lmis in weig)lt expressed as a percentage of i desiccated float ~at erial Q, 17% ~ J Weight of ether extracted material (direct weight I f'romf].ask) '"xpressed as a pci'centage of the_ air-dried float material 0. 23% Weight of ether extracted material (direct weight from flask) expressed as a percentage of the desiccated float material Q. 27% 23

1 j EXPERIMENTAL PROCEDURE PARI' D ! l I Determination of the Carbohydrate Content l 1 Treatment with Base

l 6N NaOH at Room 'rell!Perature. Whole float material which had. been washed 1 l in tap water, dilute HCl (lN), and rinsed :tn distilled water until neutral was dried in air. The dry float material (0.5485 g) was

agitated with 6N NaOH (10 ml). After 7 hr, a few drops of the clear

supernatant were spotted on a silica gel tlc plate. No charring

was observed for the spot· indicating that no c.arbohydrate had been

solubilized. After 9 days at room temperature, the float material

was quantitatively transferred onto a sintered filter, washed with

water until neutral (3X); and rinsed. with acetone (2X). The sintered

fllter and contents were dried to constant weight under an infrared

lamp. The weight of the NaOH treated float material was deterwined by

difference, and expressed as a percentage of the weight of the air

dried starting material (53%; uncorrected for moisture content or

inorganic residue).

A sample (approximately 30 mg) of the NaOH treated float material

was dissolved in cone HCl (a few ml). Small white needle-shaped

particles remained in suspension and were visible only in bright

sunlight. The solution was spotted on a tlc plate. The float material -lJ extinquished fluorescence, did not move in solvent system #1, I I 24 \ I

\ and charred with H • 2so4 l The yellovnsh colored filtrates and water washings of the NaOH l treatment were combined and concentrated on a rotovac

1,, at the front on charring. On standing, a gray precipitate appeared i 1 ' as before.

'l'he Effect of Base Treatment on Shave, --- Refract:i.ve Index and 'l'extur·e

Shape. The shape as found for the float in the live animal, is maintained

during treatment with base, whether 6N KOH for a prolonged period at

room temperature or lN KOH for 24 hr is used. On drying there :was a

slight shrinkage and resultant wrinkling, with minor distortion of

the overall shape.

Refractive Index. The whole float material, separated from the fleshy

part of the animal, was observed to be almost invisible in sea. water.

This was also observed when the float was 1rrmersed in tap water and i base solutions. No alteration is apparent when the float material is -j , kept in strong base for prolonged periods . I

I 25

Texture. Whole floats, which have been washed and dried, were smooth,

flexible and tough, being somewb.at difficult to tear with the hands. ! After treatment with base, followed by rinsing and drying, the texture I became less smooth and the w.aterial was more brittle and less pliable. · --I Purity of the Base Treated Float Material '

Millon Test. Whole float material (washed a~d dried) was placed in a

few ml of cone HN0 . The float material became light red in color. 3 Subsequent addition of cone NH 40H produced a bright orange color. However, the KOH treated float material (6N KOH, 8 days at 25°C) did not change 1 ' in color from the original dull white when subjected to the same test.

\ ~inhydrin. \!Jhole float material (washed a'1d dried) was sprayed with J fresh ninhydrin solution. The color of the heated float changed to

light purple. The KOH ( 6N, 8 days at 25°C) treated ·float matel'ial also

produced a colOI• reaction, but the color 1vas only light pink. Gluco­

sa.m:ll1G hydrochloride and N-acetylglucosa.mine were sprayed with nin-

hydrjn solution on a tlc plate. The glucosa.mine hydrochloride gave

a light pink color, but the N-acetylglucosa.mine gave no color reaction,

as expected.

~no Acid_ A.n§-lysis . Whole float material (washed and dPied) was hy­

drolyzed in 6N HCl and placed on the colwnn of aD amino acid analyzer.

the analysis indicated a high protein content (see section on protein

determination). Material which had been treated with 6N KOH for 8 days

was analyzed in the same manner, and was found to be free of amino -j acids (except for a small lysine peak). I J j 26 l DeteriT4:.nation of CaPbohydrate;Protein Ratio in Whole Float Material "I Startil]?;. Materia:),_: Whole float material, approximately 0.5 g each, l1 washed and dried in air, was put into two test tubes, and dried over I P o (65°C, in vacuo, 24 hr). The drying apparatus was allowed to I 2 5 4. cool to roorr1 temperature and the weight of the starting float material ij was quickly detennined by difference (0.5195 g and 0.4847 g). The

l float material was quantitatively transferred to two round-bottom I ,! flasks and was heated (28 hr) in refluxing lN KOH (1'7 m1) with oc­ 1 1 casional shaking. · The contents were quantitatively washed into two tared Gooch crucibles, washed with lN KOH (2X), distilled water until

neutral ( 3X) , acetone ( 3X) , and placed in a warm oven until the odor of. acetone d:l.sappeared. After drying over P o at room temperature 2 5 ~n vacuo (110 1-,:e, ?Omm Hg) the weight of the KOH treated float material

was determined by difference. The loss . of weight during KOH treat-

ment, expressed as a percentage of the weight of the washed and dried

whole float material, was 40 . 9% and 39 . 8% . Cone HCl was added to each crucible (mounted on a vacuum filter flask) to dissolve the float

material. After 2 1/2-3 hr, the dissolved float material was suction

filtered through the bottom of the crucible. After washing with

cone HCl, the crucibles and remaining sandy residue were heated at

110°C for 30 !!Lm, cooled to constant weight and the weight recorded. The weight of the sand and residue, expressed as a percentage of the

weight of the original whole float material, was 10. 7% and 10.0%. 27

Summar:,y of Results ·

Actual Weights Expressed as a percentage Recorded of the Starting Mtl.

(in grams) including excluding residue residue #1 #2 #1 #2 #1 #2 Sand and residue 0.0557 0.0489 10.7% 10.0% Loss on KOH digest 0.2123 0.1941 40.9% 39.8% 45.8% !J4.3% Carbohydrate 0.2512 0.2442 48.4% 50.2% 54.2% 55.7% (all values uncorrected for soluble inorganlc material)

Acid Hydrolysj_s of KOH Treated Float Matedal

.Method of Hydrolysis. Pyrex tubes (inner diameter 5-6 rom) approximately

.18 em long Here cleaned, dried, sealed at one end, and provided with

a narrow neck on the other end. These ampoules were cle2ned again and

dried at 1l0°C. Float material (approximately 2 mg), treated with KOH, ·and dried over P o at 65°C in vacuo for 24 hr, was placed in the tube 2 5 and the accurate weight of the float material was determined by difference.

6N HCl was added through a finely drawn glass funnel until the height

in the tube was 5-6 em. The contents were frozen in shaved dry ice

and the narrow neck of the ampoule was sealed after evacuation of the

tube (20 rom Hg). The ampoules were stored frozen until hydrolyzed.

Hydrolysj_s was carried out by immersing the ampoules in a vigorously

boiling water bath. Thr•ee ampoules were hydrolyzed 1, 2 and 3 hr.

The contents were diluted in volumetric flasks to 50 ml and colorimetric-

ally determined (described below) for maximum recovery of hexosamine. J I For storage the solutions were frozen. A second set of samples was 1 j

I \ 28 I I h,ydrolyzed and colorimetr:lca.lly determined in the same way. When obvious errors were discovered during the analysis, the runpoules l were discarded. A graph was prepared (figure 6) indicating the l .optimum hydrolysis time of 3 hr. l ~ Colorimetric Determination of D-glucosamine I 1 Equipment. The colorimetric determinations were made according to the

method of Randle and Morgan39, using a B&L Spectronic 20. The test

j tubes used were chosen for optical match by sequentially transferring l a dilute KMn0 solution to each test tube and observing the trans­ l 11 I mittance at the test frequency, 530·mu. Test tubes selected had a 1 l h sim'iJ.?.r % Transmi ttaYJce within 0 . 5%. The readings were taken from a

d:igital readout apps:ratus furnished by B,~L. The Spectror.ic 20 and

the digital readout we:'e suppJ.ied by a regulated nov power suppJ.y. ~ - i Readings were not made prior to at least 30 min warmup.

Reagents. The acetylacetone was freshly distilled before the start

of the determinations. All of' the determinations were made within

two months of the distillation and the acetylacetone was stored in

a brown bottle at r·oom temperature during this time. The ethanol

was taken directly from the container of' the supplier and was not

denatured. '!'he p-Dimethylarninobenzaldehyde had a higher m. p. than

recommended by Randle and Morgan and was not recrystallized. The

DMAB reagent solutions were stored at -l0°C in 50 ml portions. ·~~··'' .. ~~-·~···~~-"·~· ... J .... ~~-·~····~··~· ·-~-··-~~-~~·~=.. ~~~~·~~· '~'--·"'-'-'-'·"'---"-0-

'(corrected -:r~;; ~oi,~t~~~~';~t~nt'! '' +r' uncorrected for inorganic residue)! 0, '''f.l.LLl''fff''fl' , , l't 0 ~ 1 , , , 1 1 , 1 , 1 11 11 1 1 1 1 1 ; 1Ht :• ilmn::ttL 1-' 0 0 90 .....'1 1-'s:: r s 0 I <1> 0 Cll I' j ' <+ 1- _,_,1' ....'i sPl 0 .... 80 t:J ~ <1> <+ ";j ,_, <1> 0 T' ~ § i i '+ .... j;L 70 :::1 ' II> H T c+ P· ::s 1 T i TTl Lt g '' -·+1+tmr=r~TIHIIIIIII iII I! i I I I ++=4+411 ' ' 60 i Hr in 6N HCl at 100°C, 20 mm Hg ' I I I..J...... L_I I 1 I I I I I I I I I I I I I I I I I 1 1 1 , , , 1 1t 2 2t J Jt 4

Figure 6 OJ (X) PJ DETERMINATION OF OPTIMUM HYDROLYSIS CONDITIONS 29

Standards and Blanks. 'l'he standards were made up fresh from the

crystalline glucosrun:!.ne hydrochloride supplied by Pfanstiehl Labs,

Waukegan, Illinois (lot #8684). The standard was found to be free

of moisture when dried over P p at 65°C, in vacuo for 67 hr. There 5 was no weight loss for any of' three samples (approximately 1 g each)

which were dried simultaneously. The standard was submitted f'or amino

acid analysis and found to be free of' detectable amino acids (except

for a small lysine peak). The standard solutions were made up by

dissolving 0.2150 g glucosamine HCl in 50 ml distilled water in a

100 ml volumetric flask to give a O.OlM solution of' sugar. The

standard solutions were further diluted by transferring 1 ml portions

to six volumetrlc flasks (20-, 25-, 35-, 50-, 100-, ru1d 250 ml).

'l'he colorimetric results obtained for these known concentrations were

j used to prepare a graph as a basis for the determination of the ex- ~ ;~: yerimental samples, wllich were always determined simultaneously with

the prepared standards and blanks. The blanks were made by substituting

distilled water for the sugar solution, keeping all other variables

constant. Erroneous results were obtained when standards and analyses

were not carried out simultaneously. Figure 7 shoos a typical graph.

Pipets. The pipets used were calibrated by filling to the mark with

distilled water at room te~erature ru1d weighing.

Glucosamine from KOH treated float material. A sample ( 2. 5 mg) of KOH

treated float material which had been dried over P ( 65°C, in_ vacu<2_ 2o5 211 hr) was exposed to room hurnid:ity (II hr) and hydr•olyzed for 3 hr -1 l 29a l I l .

,., J soo j ~ '. : J 1 ~ "oJ .0" 400 " " : .!: ± I' i :1"' l .,a "'0 J 0 '!' 300 rl" :1 . ' I il ...."' ' I: " rl" 0 ; .. ~ "'0 I' 1- a a- .. ! 0 ... J .p ~ a ".;.l "~ .: ,, '' .p"' ..• I i " .: '- i . I • ;;: ,,.~ ' 200 "0 A .: " ' . 0 .. 0 • ;.~d .. ~- ~~~ ·~ '"

'

. ' , f' ---i-L ~~i: ri · h· ; : ; :- :- ,· '

'. i'- :: Log % Tra nsrnittance ·'I-

0 0 0 0 0 0 "'. "'. co"' co. "'<'-. <'-. '() N "'rl rl rl ...... ~·

Figure 7

Colorimetric Determination of Carbohydrate I 30 l and color:imetrically detemined as above. Extrapolation gave 93.5%. I When a raoisture factor of 5.6% (from graph) was subtracted from the j weight of the starting material the results were 99.1%. A second I sample (2.8 mg) was determined in the same way and gave 94.0% and 97.7% (after a 3.6% moisture factor for 3 hr at room humidity before

hydrolysis). Both samples are uncorrected for any inorganic or

1 amino acid impurity.

1 IR Comparison of Carbohydrate Material j Shrimp Shells. Fresh shrimp shells (species unlmown) from the Pacific

Ocean were obtained from a local restaurant within 12 hr of removal from

the frozen animal. The shells were kept chilled until cleaned several 1 j ,j days later. After being washed thoroughly in water to remove all fleshy ! rnaterlal (including taD.s and legs) , the shells were kept under i 'j 6N KOH at rooin ternperatut'e for 15 days. After being rinsed with water ~~: the shells were light piYl.k in color with white patches. The shells

were then stirred in 200 ml HCl (2N) for 5 hr at room temperature,

filtered by suction, and washed with distilled water (3X), and acetone (2X). The shell material (dried over P o , in vacuo at 65°C for 24 hr) 2 5 was chalky white in color.

Whole Float Material. Whole float material (washed, dried over P o 2 5 at 65°C, ln va_<;uo for 211 hr) was powdered in a stainless steel capsule

wi.th a stainless steel insert on a "Wig-L-Bug" and a KBr pellet

was made. The spectrum (7a,b) was compared with Spectrwn 3a,b ob-

tained jn the same way from shrimp shell, and known N-acetylglucosanrine I I 31

1 (Spectrum la,b). Spectrum 3a,b was virtually identical to Spectrum 1 7a,b l'lnile the N-acetylglucosamine (Spectrum la,b) bears a close

genera~ relationship.

KOH Treated Float Material. The ir spectrum of whole float material

which had been treated with 6N KOH at room temperature for 8 days

(Spectrum 4a,b) or lN KOH at 100°C for 24 hr (Spectrum 5a,b) were

compared. There is no detectable difference, although the pellet made

fpom the material treated for a longer time has slightly better reso-

lution. The KOH treated float material does not show a strong ab­ j I sorption at 865 cm-l which is prominent in the N-acetylglucosamine

spectrurn. A comparison with glucosarnine hydrochloride (Spectrum 2a,b) 1 !I shows a general overlap of absorption peaks, but the glucosamine hydro- l \i chlor•ide has more and sharper peal{s .

ct -D-Glucosamine hydrochloride from KOH treated Float !1aterial.

IR. Float material (approx. 0.4 g) (treated with 6N KOH for 8 days

at room temperature) was hydrolyzed in cone HCl at 65°C for 3-4 hr

to give a clear solution, evaporated to dryness on a rotovac with

methanol several times, arrd recrystallized from methanol, to give

0.25 g (63%). A KBr pellet was made from material ground in an agate

mortar. 'l'he spectrum obtained (Spectrw-n 6a,b) was identical to that

obtained r-.com the knovm glucosamine (Spectrurn 2a,b). All peal{s are

present (with no extrar1eous ones) in tl1e same proportiOYk'"ll absorption.

TLC. A few drops of the ct-fi-glucosamine 11ydroch1oride prepared as

above, wer'e chromatographed on a tlc plate with known glucosamine 32

I! hydrochloride aJld N-acetylglucosamine,'with solvent system #2 and

charred. The Rf of N-acetylglucosamine was 0. 41, while that of the

glucosamine hydrochloride and the experimental sample were,identical at 0.32.

This solvent resolves the experimental sample into one major and three , 1 very minor fractions • The minor fractions may be a product of the I hydrolysis conditions or may indicate the presence of other similar ., " sugars in minor amounts (figure 8).

1! Elemental Analysis of Carbohydrate 1.,

For. elemental analysis, a sample of float material (treated:

lN KOH, l00°C, 24 hr) was carefully selected to be free of visible

sand, suspended in 6N HCl and deaerated in vacuo (20 min), filtered, ' ------' washed with disti1led water to neutrality, and with acetone (3X).

It was dried at 65°C in high vacuum to constant weight. The cal­

culated values are based on an expected N-acetylglucosam:l.ne polymer

of inf:l.n:l.te length (m.w. 203). 1 I Calc. C,47.29; H, 6.40; N, 6.90; O, 39.41; CH co, 21.18. 3 1 Found C, 47.28; H, 6.43; N, 6.95; O, 39.48; CH co, 21. 53. 3

Amino Acid Analyzer Results

Whole float material which had been washed and dried, float j j 1 l j I 32a l I l '1 I ~·------~------~------(front) N-acetylglucosamine

glucosainine HCl

experimental

. ~. (J'

' ' ·.. ~ I !

1------•------....------(start)

Figure 8

Thin Layer Chromatographic Comparison of the Acid Hydrolysate of KOH Treated Float Material with N-acetylglucosamine and glucosamine hydrochloride 33

material whiC'-h had been treated with 6N KOH for 8 days, and known samples of glucosamine hydrochloride and N-acetylglucosamine were ana1yzed on a Bio-cal 201 Amino Acid Analyzer. The peaks indicated a large concentration of glucosamine in the wnole float material

and in the hydrolysate of the KOH treated float material. When

quantitative ca1culations of glucosamine content were ~e from the chromatograms using the known ·N-acetylglucosamine as the standard, the· KOH treated material gave a 96.5% recovery. lfuole float material gave 33.8% N-acetylglucosamine content. A correction factor (15%) for moisture increased this to 38. 5%. A second .sample gave 40.6% and 49.6% respective1y. When the found carbohydrate content was ca1culated as a percentage of the total protein and carbohydrate found, values of. 60_. 4% and 59.4% were obtai_11ed.

J --i· '~.

I 1 ! i i l j \ 34

Surrnnary of Carbohydrate Determination

By Difference 1st KOH treatment 53% 2nd KOH treatment 54.2% 55.7% Direct Determination (Amino Acid Analyzer)

~fuole float material lst determination (inaccurate standard) 33.8% Subtracting 15% moisture factor 38.5% 2nd determination 40.6% Subtracting 15% moisture factor 49.6%

KOH Treated Float Il'iaterial -~Recovery as a percentage of known standard· hYaro1.yzed silfuitaneously) Carb_2hydra.te Percentage (Carbohydrate as a percentage of total protein and 60 .4.% carbohydrate found) 59.4%

Colorirnetric Determination of Hexosamine Content 1st determination KOH treated material (uncorrected for any inorganic residue) 93.5% corrected for 5.6% moisture content 99.1% 2nd determination 94.0% corrected for 3. 6% moisture content 97-7%

Elemental J\nalysi~ Analysis matches for c H No (203). 8 13 5 Acetyl group analysis indicates complete acetylation. I -lI I 35 I I j IR Whole float material matches known chitin. Acid hydrolysis product of KOH treated material matches glucosarnine hydrochloride •

TLC Acid hydrolysate of KOH treated material and glucosarnine hydrochloride have identical Rf values. Il EXPERIMENTAL PROCEDURE I PARTE Determination of Protein Content

Millon Test

1 Whole Float. Approximately 100 rng float material (washed and dried)

was treated with l rnl cone HN0 . The float material became red in I 3 color and changed to bright orange when 3 rnl NH40H was added. 1 1 KOH ~reated Float. Float material (washed and dried) treated with

l KOH.(lN, l00°C for 24 hr; 6N, room temperature for 7 days) was

washed to neutrality :tn water, and acetone and clried :tn air. Tr>eatrnent

with HN0 and NllLeH as before did not change the color. 3

Ninhydrin

v~ole Float. Whole float material (washed and dried) was sprayed with

ninhydrin solution. The material was placed in an oven (l05°C) for

10 m:tn during whi.ch a purple--pink color developed.

------KOH 'I'reated Material. Float material washed and dried and treated with KOH (lN, 100°C for 24 hr) was sprayed with ninhydrin. A light pink

color developed.

Ashing

Whole float material (washed and dried) was ashed in a crucible

to whiteness. During the r,shing a distinct odor similar to that of

burning hair was detected. 37 \ 1 I I ~lemental Analssis for Phosphorus Whole float. material (washed and dried) was analyzed for c, H,

O, N, and F. Found: C = 41.3%; H = 6.56%; 0 ·= 43.15%; N = 8.99%;

P = 0.0% (less than 0.2%).

Sulfur

Sodium Fusion. 11Jhole float material (100 mg; washed and dried) was

1 heated to redness with metallic sodium. The hot test tube was shattered

lI in 20 ml distilled water. The filtrate (2 ml) was treated with 10 drops j of 5% lead acetate solution. A ver~ faint black precipitate was ob­ l served. Float mater-ial (washed and dried; tr·eated with KOH as before) ! ! gp.ve i.n con1pcu;ison no black precipitate. ' ---i l . Formalin Preserved Float ] A velella specimen which had been kept in a cone formaldehyde solution

for a number of years was examined periodically during treatment

with 6N KOH (25°C for 25 days). In bright sunlight flat portions of

the float were obser"red separating from the vertical component and

smaller portions were observed separating from the horizontal component.

The materials were not analyzed . The formalin treated floats showed pro-

nounced da.rkenir.g and shrinkage when washed and dried after KOH treatment.

,l 'I 1 l 38

Percentage of Protein by Difference

KOH Treatment. The loss of weight which occUITed when whole float

material was treated with KOH was determined as described in the

carbohydrate determination. 'I'his loss (from two determinations) .

was 45.8% and 44.3%. The filtrate did not contain any material that

could be charred on a tlc plate. The KOH treated float· material

was examined on a Bio-cal 201 Amino Acid Analyzer and found to be

virtually free of amino acids, with the exception of a small amount

of lysine, which still persists in the KOH treated material. The

glucosamine hydrochlorideand N-acetylglucosamine standards were tested

in the same way and were found to contain also detectable amounts

of lys:tne. A small peak tentatively identified as galactosamine also

occurs in all of the chromatograms .

Percentage Protein By Direct Method (Amino Acid .Jlf.!§:J,yzer)

~ydrolysis. Float material (washed and dried in air) and redistilled

6N HCl (1 ml/1 rng) were frozen in a glass tube at -70°C. The twJe was evacuated (less than 0.1 rnrn Hg) and sealed. The contents were

hydrolyzed at 105° (16 hr). The clear solution was transferred to a cruc:tble, dried over P o and KOH (less tl1an 0.1 rnm Hg) , redissolved 2 5 in water, transferred to a 10 rnl volumetric flask and broug_l)t with

buffer solution to pH 2.2.

~-~)Jr:?i~. For the analysis an Amino Acid Analyzer, BC 201 - Biocal I Instrument, Mlinchen, West Germany, was used. The one-column technique -1 i with suitable arra11€;ed buffer changes gave chromatograms which were I compared with known mixtures of amino adds. 39

Evaluation of the Chromatograms. For the calculation. of the amino

acid content of the hydrolysis, the method of CREMER9 was usedwhich 46 is supposed to be the best choice for manual evaluation • The peak­ height. (h) and the peak-diameter at the half height (1/2 b) were measured

with a calibrated magni:t'ying lens. Then the F-values were calculated

by the formula F = h·l/2b. These values were compared with the lmown areas of standard chromatograms and the arnount of· amino acids 'I I calculated. The results for the mole ratios (Leu = 1) are given 'I I in Table 1. N-acetylglucosamine was hydrolyzed and determined in the same manner and used as a standard for the glucosamine calculations. I The carbohydrate results are g.i.ven in Table la.

Table 1 Amino Acid Analyzer Results

lj v... Au.:tno Acid ~~: Nanomoles NanogriUu~~of JR ~'til -~- M.!il ~oifial ---- __ Foun.1_.~. _.(_i;}lousl'J:ld ·U&..I.! e u ··i * • ~ Aspartic Acid ~1312.2 'l14 J2,0 J, 5 l'> lj. 115 460 h.;;n ~. 36.1 '·9 4 tt ' Threonine ~ !z6·o 25.6 23 ·3.2 .. 3? 3 101 22A 2 .o 2.8 . 1 303 •n• Serine 314 27.4 3·9 1,? lj. 87 348 -~ 3ll 27.0 4 •LLR ' Glutamic Acid 1 585 75.6 7.,2 7 129 903 2 644 8),0 8.1 8 JQ:J? Proline 396 ?7.9 6 "·9 5 97 485 . ~ b.01 9 .5 6.2 6 582 Glycine 1 90.8 5.16 1·1 1 57 57 2 12h 7.19 1·57 1 '>? Alanine 1 125.2 8.95 8.46 1·55 1 71 71 2 11R 1o47 1 ?1 Valine )62 35.8 4 f'f·5 s, ) 5 99 495 ~ 41? 0,7 .2 lLO< Isoleucine 1 10.5 11.8 l;J 1 113 113 2 CJ2 10.39 1.16 l 111 Leucine 1 81 9·15 8.98 1 1 llJ 113 2 '7CJ." 1 1 Tyrosine 1 112 18.3 4 6 ll·'< 1 103 163 "' 2 8A.1 . 1 .3 loll 1 16• Phenylalanine 1 124·. 9 18,3 ~ '< 1«7 29/.f 2 'RL 23. 6 2.0 2 294· lzy'sine '71 60.4-66.9 ,5•~ 0 l>:tl 7<>8 ~ '>2'1 6.6 6 768 Arginine 1 166.5 26,0 -z;o- "- 15b 312 2 1?2 26.8 2.16 2 31£__ Half Cystine 1 102 J.U?V -;r-.-,::;--- -,;u, 2 264 26.7 3·3 " 3 JVO 01 R -1 Totals 3733 . 442.8 44 5191 48 -- 6029 -- 4088 - 526.7 "\.. Calculated Minimum M. vi. 40

Table la Carbohydrate Found

N-acetyl- 1 3.320 675 glucosamine 2 .3800 771 Any trytophan present was degraded 1n the acidic hydrolysis. Histidine and methionine were not present in the sample.

Sample #1 = 2.0 mg for hydrolysis #2 = 1.9 mg for hydrolysis A moisture factor of 15% was subtracted from the starting weights and the results calculated for both the dried and undried materi&l. The results of two determinations, expressed as a percentage of the dried and undried starting material were:

'I'able 2 Protein Found in Amino Acid Analysis -- Sample #1 #2 - Room Humidity 22.1% 26 • .3%

15% Moisture 25,0% Factor Subtracted .31.0%

The total protein and carbohydrate found were summed, and their relative percentages calculated. The results were:

Table 3 Calculation of Protein and Carbohydrate Ratio

~. ~TFound __if Of~.t~-...- 1 o.Iii}"J. .39. % J.:.l. ITot~l Protein 2 0,1)2? 40e6~ -· 1 0.6'75 6o.4% . ~'otal _carbohydrate I 2 .. 0. 7'?1 59.1~% j Total l''ound 1 1.118 100,0% 2 1.298 100.0% - ~-· ar I ---'------I 41

The recovery of the starting material after hydrolysis was calculated for both dry and room humidity conditions. The results were:

Table 3a Total Protein and· Carbohydrate Found as % of Starting Weight Sample Weight Total '!G Of Starting Weight (air dry) Found Air Dry 15% Moisture Factor Applied 1 2,0 mg 1.118 mg 55.9% 65.7% ----- 2 1.9 mg 1.298 mg 68,2% 80.4% -- J'ilole ratios for the total amino acids found and the total N-acetyl- glucosamine found were calculated for both samples :

Table 3b Mole Ratio of Total Amino Acids to Carbohydrate total amino N-acety~- mole I sam-· acids .found gluco_sam~ne ------~-- -- ·nre G1anomoi esT ----(nanomoles) . .. ratlo ~ ~--·-· 1---.- ---1-'--- JJ20 1.12 11 f.-~ 3733 2 I 1>088 ]800 1.oa·-;1 1 --~------Chromatograms of KOH treated float -~terial were examined for amino acid content. Three peaks were present in the glucosamine-arrmonia region and labeled #1, #2, and #3 in order of elution pending identifi- cation. The peaks were also present in the commercially prepared N- acetylglucosamine standard and glucosamine hydrochloride. Muramic acid prepared in this laboratory 1~as free of peaks #1, #2, and #3, but contained a new peal< in the same region. Peak #3 was tentatively identified as lysine; and though peaks #2 and #3 were unidentified, galactosamine and hydroxy lysine were considered possibilities. The peak areas were determined as before. Mole ratios for lysine : N­ acetylglucosamine gave an average of one lysine for 8,000 carbohy­ drate monomers. The complete results were tabulated (Table 4) . =·'- '-"-~·~··~·

unknown peaks lysine N-acetyl- I __glucosamine mole test #1 #2 #3 I nano- micro- micro- sample 2 2 2 ratio mm mm mm2 I mm moles grams· moles vertical component 9.0 4 8.8 I Zj~xso= 1.02 1,850 9.1 1• 8900 (KOH treated) horizontal component 11.7. 6.7 11 71}00 (KOH treated) 4 7·9 kjgx50= 0.91 1,360 whole float 1 9.42 (KOH treated) 20,6 4 11.5 ~jtx50= 1.32 1,930 1• 7140 N-acetylglucosamine . 10.2 50 11 8400 (standard) 15.4 4 10.2 I+Jbx = 1.17 2,000 9.85

glucosamine x50= 1,880 9.25 11 8170 hydrochloride 9.3 3.5 9.9 M3 1·13 I ;; avg. 1.37 avg. 11 8000

Table 4

Lysine Residue in Standards and KOH Treated Material

£\.)""' 43

\ The mole.ratios of lysine to other amino acids present were calculated from Table 1. Samples #1 and #2 averaged 5. 5 and 6 .1 lysine residues I respectively, and 5.8 when combined. The complete results were: Table 5 Ratios of Lysine to Other Arnino Acids l Amino Acid sam- MoTes Ly~ine( ~oJ.e l s ple Other AmJ.no Acl.d Leucine 1 5.8 2 6,6 Isoleucine 1 4.5 2 z; .. 7 Tyrosine 1 4.2 2 '\,9 Glycine 1 5.2 2 4.2 PhenylalanJ.ne 1 '('.~ "' 2 2 6.5 Arg2mne 1 5:7 ·--· - .. = 2 2· - 6;1 'l.'hreonJ.ne l. 5.4 I -- 3 2 6.2 · Aspara.g:ms 1 o:·o-- -- = 4· 2 6.7 ·- Average 5.5 6.1 Grand Average (of all 16) 5.8

The lysine : carbohydrate mole ratios for whole float material were

1 : 7.1 and l : 7.3 for samples #1 and #2 respectively (Table 6).

Table 6 Mole Ratio of Lysine to Carbohydrate _j lysine N-acetyl- I found glucosamine mole I sample found ratio (nanomo] es) (nanomoles) l -- 1 471 3,)20 1 I 7.1 -----2 523 ),800 -·----11 7.J 44

.i I Lysine : carbohydrate mole ratios expected for the minimum molecular 1 protein weights determined in the amino acid analysis (Table 1) and p1•otein : carbohydrate weight distributions suggested by the quanti­ I tative results were calculated. The molecular weight of an hypothetical i protein with lysine links to six octasaccharides (figure 9) was in'­ l cluded for comparison. Values of 1 : 5.2 to 1 : 8.6 were obtained. i The complete results were: 1 Table 6a Calculated Mole Ratio of Lysine to Carbohydrate

lysine to carbohydrate j mole ratios l protein to carbohydrate m.w • of protein we lght _ratios__ ,______l----·-- ~; ~ ~ :~o-.. __·r~-6-_ ~-:c-~-~---~:. _ ------j 5 0 6 ~- 1 r-·--Tr5T55___ 11 5.2 11 6.o --11 7.0 ' l 41.5L~ I 58.46 11 6,0 11 7.0 11 8.0 ' --1 /j.o I 6o 11 6. 9 11 - '7. 4 11 8. 6 i '--~------·---- ~ A similar calculation for the protein : carbohydrate mole ratios gave values of 31.4 - 51'..6 N-acetylglucosaminyl units per protein.

~he complete results were:

Table 7 Mole Ratio of Protein to Carbohydrate

protein to carbohydrate · mole ratios r------1- protein to carbohydrate -~-~{, of_m;:otein__ -i weight ratios ~6o 6~go 6a~; 11 31.4 11 36.1 lt 41 • 6 ------···-·----45 I 55 41.54 I 58.46 1 I 36 , 0 lt 41 • 7 11 48 o 0 -----·~--··-----,.·---····- IW I 60 '1".-3-8:1+ -;-;--44. 3 11 51 • 6 ·--- S1.lllli1Ery of Protein Determination

N:irl.hycli'in Test Whole Float positive. KOH treated float material positive j (diminished; with color change) l 1 Millon Test l Whole Float positive KOH treated float material negative 1

1 Ash~ Definite odor of burning protein 1 j l ~ %ProteiU~ifference 1' Loss on KOH treatment 47.0% ---j t1 U IT II ~ . (quantitative) 45.8% 44.3%

%Protein by Djrect Determinatjon (Amino Acid Analysis) Found in air dried sample 22.1% 26.3%

15% moisture factor applied 25.0% (uncorrected for loss in hycli'olysis) 31.0% As a percentage of the total protein and carbohycli'ate found 39.6% 40.6% (uncorrected for loss of amino acids in hydrolysis)

-~

i lJ6 I CHAPI'ER 3 1 I Brief Swnmary of Results

Moisture Content l Whole Float ==--- Over Cac1 ( 48 hr, 60°C in vac_g£) ~ 2 10.]-% Over P (67 hr, 65° C in vacuo) 2o5 14.5% KOH_ Treated Float Mat~r:ial Over. (75 hr> 65°C vacuo) r2o5 if!. 14.1% 14.1% 15.3%

~!------~------~------~------~ i ' I1J.~~9]~9__GOQ~E!t. ~~l2.§Xt£aC~ 69.0%

Whol~ Float_ !'2_aterial (Dry :B-sh) Washed; visible sand present 2.8%, 3.0%, 3.7% Washed, blended and sorted; free of visible sand- 45ooc ash 0.8% 550°C ash 0.5% After HF treatment 1.1%

VJ!Jolf:. P'lo~i !1ater:lal. _{VIet Ashl_ Cone IJN0 /!IC10 3 4 2.7% Reweigh after 12 hr 2.8% After treatment VIi th cone IJN0 3 2.9% After treatment VIi th cone !IF 2.6% I Subtractlng 14% moisture factor 3.1% -] I J --KOH ~reated Float------Material lN KOH (24 hr at 100°C) (Oxalate positive for calcium) 1.8%, 3.6%, 3.2% 6N KOH (3 days at 100°C) 3.3% 3.4% (red ash) 7.7% Desiccated .KOH tre8.ted. float material 4.4% l.,•I ~

1 Lipid Content ;Lst_ DeterrJg_nation ll I By Difference 0.14% ' . App).yi.ng 14.5% rnolsture factor 0.17%

2nd Deterrnin~tion !?x. Dlfference

Alr Dry 0.15% Deslccated (over P , 60°C 3 hr, 20 rnm Hg) 2o5 0.17%

Air dry o. 23rr. Desiccated (as above) 0.27% 118

parbohydrate Content

vlhole Float

1st Determination room h\l!P.idity/6N NaOH 9 days, 25°C (dried tmder infrared lamp to constant weight) 53.0%

:<'J.1d J~EO-tEO!'!!fi-nation 24 hT' at over· P 2o5 at . 65°C for 24 hr in vacuo/lN KOH 100°C, washed ~~d dried as before. 54.2% 55.7% Amino Acid Analyzer Determination B'ound jn air dried sample 33.8% 38.5% 15% moisture factor appHed 40.6% 119.6% As a percencage of total material found 60.lf% .59. II%

Protejn Content i fercent§g£ Protein by Difference (KOH treatment) I nonquantitative 47.0% I quantitative 45.8% 1 411.3% I Direct .Meas,yement (Amino ~id J\nalysis) l F'ound in air dried sample 22.1% 26.3% 15% moisture factor applied 25.0% 31.0% As a percentage of total material found 39.6% 40.6% lJ9

.I 1 Composition of Velella lata Float Collected From The Beach l As Suggested By The Analysis_

! Moisture content (at room humidity) 14-15 wt % l' As %of Dry Weight

InorgeJnic Content 3-4 wt % ij Lipid II 0.2-0;3 wt % ,,I ,, Carbohydrate " wt % 1 55-58 I Protein 11 42-45 wt % I Protein/Carbohydrate 45/55 wt % l l l l ! J 1 ---,1 l ~- ) I I l \ CHAPTER 4

DISCUSSION OF RESULTS PARI' A

-1 ! Moisture Content 1 The float material is saturated with water in the intact a!'.ima.l. l l The removal of water represents a departure from the physiological condition. When the moisture content at room humidity was determined

the amount of water in a saturated float was not investigated since

it did not seem to be gerruane to the analysis, For further work,

it would be interesting to examine the rate and amount of water ex-

change out to the satu:cation point.

The determination of moisture content at room hum:t.dity allowed

the application of a moistu:ce factor to material which had never been

dried. The average humidity in the laboratory was not determined

and no gmph was prepared showing the moisture factor at different

levels of humidity, pressure and temperature. To this extent the

moisture factor is unreliable, When whole float material was dried over cac1 and P o a difference of 5% was noticed and thereafter all 2 2 5 drying was done over P o . The loss of weight on drying of whole 2 5 float material was compared with that of' the KOH treated material and

found to be the same wi.thin 1%. This seems to indicate that the re-

moved protein absorbs water to approximately the same extent as the

carbohydrate. 51 j j The resorption of water was studied to give some indication of

l how fast the dry material regained the lost water. A rapid water ! uptake was not found and no special weighing or handling was required. l The resorption of approximately 10% of the lost water in one hr in­ dicated that appreciable errors could be avoided by quickly weighing I the material. The delay in resorption indicated that, once dried, J the float material requires several days before it can be treated as

1 equilibrated with the room humidity. The increased drying over P o relative to CaC1 raises the I 2 5 2 question as to whether all moisture was completely removed by the P o 1 2 5 1 either. The recovery of carbohydrate was over 96% in the color:!Jnetric ___ J~ ______detennimtions_under_Pptii11J.JlllJ;:RDQ;l,i;ion§, _ I_n_th~_ill!L1J10__ ac:_~d,_8.!18o},Js_:L§______I' ~- there was a small amino acid irnpm•ity (lysine), an undetermiiJed peak (possibly galactosamine or hydroxylysine), plus an undetermined I inorganic residue. These_ factors, plus- any loss •· of the_ carbohydrat_e 1 in the hydrolysis, suggest that the residual water is less than 1-2% .1 when the material is dried over P o in ·vacuo at 6'5°C for 24 b.r. 2 5 l1 Float material which has· been washed equilibrates on drying in air overnight. This time is shortened by washing in acetone. The

weight-attained remains reasonabl\)r constant over a period of time and

suggests a special relationship between the water and the polymeric

matrix. ltihen the resorption of water is calculated for the KOH-treated

material as a ratio of moles of water resorbed to moles of polymeric

matrix (using the weight of the N-acetylglucosaminyl residue-203-

for the calculation) lt is found that the ratio is between 1-2 moles

water to 1 mole of monomer. ltihere the ratio goes from 0 : 1 to 1 : 1,

the water 11ptake appears linear, but thereafter the rate of resorption 52

decreases sharply. This may indicate that ~chitin, described as the monohydrate by Dweltz1P, may. give the ex diffraction when completely

dry. Very accurate determinations of moisture content require that I the inorganic content be determined and corrected for by ashing the l --i dried sample. Unfortunately there are difficulties in the drying fonn of calcium, and coupled with the unknown silicon relationship, ! the question of free calcium versus.the carbonate, the presence of other unknown inorganic material, and the difficulty of handling the large sample sizes required for accuracy, it is doubtful that a significant improvement in the moisture values would result. The

acceptance of a 14-15% moisture content in both the untreated and KOH-treated float material under ordinary conditions of temperature ------and humidity seenis t:o b~ justified by the data. ·A more detailed investigation of the dry:i11g and resorption rates m:ight g;ive some in::l.ication of how the water is attached to the polymer.

I 1 1 I I 1 53

DISCUSSION OF RESU'JLTS lI PARr B

Inorganic Content

--~ The Velella float material for tr~s study was collected on the 1 beaches of Northern· California and Oregon over the past two years, I The removal of sand, which adheres tenaciously to the dr-led float, 1 represents a challenge which was not completely met . Sand grains j come in all sizes, and the assumption that the sand could be removed I 1 manually by visual inspection proved fallacious. The fact that the I 1 horizontal portion of the float is a "double" merribrane with enclosed j ---- -air-spaces- compounds -the--problem.- -When--sandy-float material-is-washed------I carefully by hand, one float at a tirne under a running tap, the re- 'J l'• w.airli.ng sand content can easily amount to 10-15% of the total weight. ~ -. With careful selection of sand-free material this can be reduced I I to 2-3%. The best sample, which wat; blended in a Waring blender, I was reduced to 0. 8% , but the blending may have produced 1.mknown j fractionations and it was not used as a general cleaning technique. '! The residue after ashing was treated with cone HF to determine

the silicon content of one sample by converting the silicon to gaseous

silicon tetrafluoride. The sample increased in weight almost 100% and leaves the question undecided. Such an increase can be partially

accounted for if the anion is converted from oxide to fluoride. The

small sample size (4.5 mg) of the ash makes possible an appreciable -1 error in the weighings . I Does Velella u.Se silicon as a· component of the float structure?

The present collection method renders this question unanswerable,

as a silicon compound (e.g. Si0 ) may be removed as extraneous material 2 in the cleaning method that was used. In this connection one may recall that silicon is used for structural purposes in silicoflagellidae,

sponges, and radiolaria, and is essential for the synthesis of connective tissue of the chick5. This means that a preferred metho:i of collection would be to scoop the live Velella animals from the open sea and

freeze them u~til a careful separation of the float could be made in

the laboratory. J Sea water contains a high concentration of dissolved salts, and I .. to some degree the Ve1e1la exod<9ri111s Sf:rves as a sernipi'J;rmeable bi3.J:>rier l l behind which inorganic material may acclllnulate. The float, once freed ~ --l from the protection of the intact animal, 1/lould be expected to. eq11ili- a l ii. 1! brate with the salt concentration. This expectation is tEJmpered by

the observation that the external coverings of many marine anima!_s do not appear to be protected from the sea and do not appear to lose their inorganic content. These materials conta.in a high calcium content in a "chitinous" matrix similar• to that found for Velella, and the

extrapolation might be made that the inorganic content is not diluted appreciably by exposure to the sea and to rain. A value of 69% for the inorganic content of filtered wash water suggests an appreciable inorganic content in vivo. A better answer awaits improved collection technique. Several attempts were made to separate the water and acid soluble \ 55

components from the ash. The small quantities usec:l and the minor differences noted did not allow any conclusions other than that pro­ duced by other methodS, i.e. that the inorganic content is a very small percentage of the total weight.

It was of li~erest to determine if the residual inorganic material survived the KOH treatment or was removed with the protein. Three simultaneous determinations were made in the same way on KOH treated samples and calcium was precipitated with ammoniUm oxalate by the 26 method described by Kalthoff and Sande11 , indicating that KOH treatment does not rewDve all of the inorganic material.

Treatment with acid would be expected to dissolve any calcium that was origi.YJally present as the carbonate. Hydrolysis of the float material with strong acids did not result in a completely clear solution however, and this acid insoluble residue remains a nwster

The resl.due con:otitutes an in.significa11t amount of the total weight and is only mentioned as a suggestion for further work. 1tlhite needle- shaped particles remain suspended when the KOH treated material is dissolved in concentrated HCl, gray suspension remains after hot concentrated HN0 -HcL0 ox:i.dation, and red particles appear on pro­ 3 4 longed treatment with concentrated HCl. Values of about 4% inorganic residue for the whole float and

1% for the KOH-treated material were found by the Beller Microanalyt- ical Laboratory. The primary interest in the inorganic material was to try to elim:i.nate :i.t entirely so that j_t did not obscure the protein and 56

l carbohydrate dete:rnrlnation, but it is interesting to consider the

l possible function of the minute amounts which are present. The finding of 0. 8% inorganic material seems woefully insignificant, but a mole

I of calcium (m.w. 40) represents only 0.67% of the weight of a protein J l with a molecular weight of 6,000 when the mole ratios are 1 : 1.

!' A carbohydrate polymer of eight N-acetylglucosaminyl resi.dues (m.w. 203) has a molecular weight of about 1640. and could coordinate with a divalent

cation (four ligands) in a mole rati.o of 4 : 1 and the cation could still be well under 1% of the total weight.

The presence of aequorin in other jellyfish raises interesting l questions regarding its possible biological function. Aequorin is . t a protein which specifically bioluminesces in the presence of free fl 48 l calcium ion , The energy is released at contact, so that the aequorin- · '·; ~ ] calciu'll corrplex may be assuJT£d to be a lower energy and thennodynamicaJ.ly

<;~ I more stable confonnation"j. Phosphorescence is a common observation of disturbed marine animals. Does physical disturbance of the anirnal

(811d the matrix) dislodge free calcium, which then triggers the stored

energy of aequorin-like proteins? If so, how is the calcium sub­

sequently freed? Is light requi.red to free the calcium, which might

then be available for incorporation into the matrix? It seems clear

that there are other alternatives to acceptJ:ng the viev; that the

calcium is present in the matrix only as the carbonate,

What can be said with confidence regarding the inorg8l1ic material?

The difficulty of removing the s8l1d c8l1 be overcome with irrproved

collection techniques, and the loss of soluble material minimized, 57

so that reliable iiJformation regarding the in vivo condition can be accumulated. Calcium is certainly present, and so tenaciously as to arouse suspicions of coordination in the matrix. Material collected from the beach has a residual inorganic content which can be lowered I to 3..,4% with careful washing. Blending and meticulous sorting will l give material with less than 1% residue. The possibility of determining the specificity of calcium for the extracellular nntrix is suggested. 1 I DISCUSSION OF RESULTS PARr C

Lipid Content ---1 The first attempts to determine if a significant ether soluble l fraction existed in the float material gave inconclusive results. 1 The possibility that the washing procedure was extracting the lipid fraction was considered. A large portion of unwashed sail material

was cleaned of debris and free sand and washed extensively in a minimum

amount of diethyl ether several times. The ether was subsequently l evaporated to dryness at room temperature. A thin film of oily residue I " rema1ned with the sHght odor of cod liver oil, indi.cating either a l" J i!'f. j negligible lipid fraction, or a tightly bound one. -----; j Carefully conducteo. quant1tative extraction in a soxhlet apparatus j gave results under 0.2% in two determinations.

1 When a portion of an animal is removed for investigation, there is always the question of surface contamination. The float material is

non--cellular, but occurs in the animal immediately adjacent to actively

synthesizing cell membranes. Is it possible that the float material

absorbs a portion of the lipid material found in membranes when the

animal dries on the beach? The exposure to the sea and ra.in might

not remove lipid material which is only sllghtly soluble in water. I 1 Is the lipid an i.ntegral part of the float in the live animal, asso- I i ciated with the protein or carbohydrate, or· both? These questions .,I remain unanswered for the moment . When the protein is removed in the j KOH treatment an oily film is observed on the wash water, indicating I 59

. the necessity for washl.i'Jg with acetone. This makes i.t probable that the small lipid content is removed with the protein. 'l'he distinction between "ether soluble" and "lipid" is signi- ficant. The presence of a large lipid of 16-18 carbons represents a mole ratio of about 1 : 1 to the protein. A small ether soluble molecule, with 2-3 carbons, would have a mole ratio of about 1 : 6 to the car­ bob..ydrate. In both cases a significant role can be seen for such a molecule in the matrix, and it should not be altogether forgotten. The identification of the lipid would be helpful in resolving its role relative to the matrix. The float material was dried after extraction to .prevent possible

loss during the drying process. It would be interesting to determine the voJ.atility of the ether soluble component, sirice it mlt;ht be included in, or be related to, the moic.ture loss. DISCUSSION OF RESUI~S PARI' D

Carbohydrate Content

\v.hen Rudall examined a wide variety of invertebrate connective

tissues using x-ray techniques, he obtained a characteristic dif­ 40 fraction pattern by which he defined "chitinous" material . Although

he treated the float material with 5% KOH at 100°C for 24 hr prior

to his analysis to free it of protein he failed to get such a "chit­

inous" pattern from Velella spirans float material and suggested that

additional purification would yield the characteristic diagram.

Float material which was so treated in this work was submitted to

amino acid anaJ.ys:i.s and found to be at least 96.5% pure N-acetyl-

glucosarni.ne. The same material when hydrolyzed and determined color'-

irnetrically again gave 96. 5% recover-y of N-acetylglucosamine. The

determinations were made for glucosamine after hydrolysis but the

elemental analysis of this material indicates that it is completely i N-acetyJ.ated. 'l'he major impurity appears to be lysine, which remains

1 after ba.se treatment and survives subsequent acid hydrolysis, and j possibly a minor amount of galactosamine and hydroxylysine which

occurs on the amino acid analyzer chromatograms. Rather than purity,

heat j_s more likely to be responsible for the x-ray results. The

longer treatment (6N KOH, 6-8 days at 25°C) would probably give the

usual diagrams. Thin layer chromatography in a variety of solvent

systems indicated that the carbohydrate was pure, but when solvent

#2 was tried, late in the analysis, it was discovered that a separation 61

into one major arid three minor components occurred. These findings

do not necessarily contradict high recovery of N-acetylglucosamine

and the question of other sugars being present is still open. No

muramic acid was found when amino acid analyzer chromatograms of known ·

l!lLlram:tc acid 'llere compared with chromatograms of Velella float material.

The identification of the monomer rests upon: The ir comparisons

with known chitin, N-acetylglucosamine and glucosamine hydrochloride,

the colorimetric tests which are specific for hexosamines, the amino

acid analyzer results, the elemental analysis, and tlc comparisons

with !mown glucosam:ine hydrochloride and N-acetylglucosamine. Melting l points matched for chitin and glucosamine hydrochloride, but were not l considered significant for identification. I The use of enzymes to degrade the polymer (chitinase and chito- J biase) would give a direct recovery of the monomer without the dea~

cety1ation which occurs with acid hydrolysis. These enzymes were.

not a'!ailable for the present study:

The high recovery of the N-acetyl group in the elemental analysis

I. indicates that the 6N KOH treatment (7-9 days at room temperature) ! does not appreciably deacetylate the polymer and that the carbohydrate

is probably completely N-acetylated·in viv~.

1'he elemental analysis on the whole float indicates that phos-

phorus is not present. 1'he metabolic pathways found for carbohydrate biosynthesis19 include phosphorylated intermediates. By the time the

carbor~drates are incorporated into the polymeric framework of the

Velella float these intermediates are probably absent, unless lost in 62 j the collection and wash:Lng methods used. The polymeric matrix is organized in an unknown way. The possi- l bility that the polymer is one long infinite chain is unappealing, l j and biologically illogical, since the float appears to be synthesized and la.id down as a topologically continuous membrane on three surfaces

s:!multaneously. The association of the monomer into subunits is a

more attractive concept, and is supported by suggestions that a polymer 8 monomers long is in evidence2, 42 .

Some evidence on the levels of organization was offered by ob-

serving the float material during the acid hydrolysis. When KOH

treated material was hydrolyzed in 6N HCl in a large test tube at

100°C the initial condition after shaking was the sedimentation of

the entire mass. After seve:c"al hours and intermittent shaking the

ma..'ls distributed itself evenly throughout the test tube. S

which were free in. the solution did not fall to the bottom of the ~ -- tube. After another hour of heating at 100° the sand grains slowly tumbled down through the matrix to the bottom of the tube. This

change was reasonably abrupt, suggesting a change in the structure.

After more heating and several hours la.ter, sand grains brought to the top fell without interruption to the bottom of the tube. Placing

the tube in. an oven at 125°C for ten minutes completely cleared the

solution. Browning occurred during the hydrolysis and later the

solution assumed a distinct dark brown color. After hydrolysis, when

the tube was held against bright sunlight, small needle-shaped par-

ticles were visible which appeared crystalline. These five abrupt 63

changes may correspond to the five levels of cellulose organization32 ,

;I'he amino acid analyzer results suggest a protein of 44-60 amino

acids with a m.w. of 5,000-7,000. The mole ratios .for theN-acetyl-

glucosaminyl monomers give values suggesting 31-52 monomers per protein. J If the carbohydrate is linked to every protein by six linkages, as the 41 42 x-ray and electron micrographs • seem to show, then an oligosaccha­ ride length of 5-9 monomers is indicated. The acid hydrolysates · 2 produce a seven monomer unit (which is better evidence for eight

since the end sugar would be expected to degrade in the hydrolysis) 41 42 and the y -chitin studies • indicate that eight is the proper

number. If the eight monomer unit is used with the weight distri-

butions and the m.w. findings, a protein Nith a m.N. of 6,800-7,000

is required (if there are six octasaccharides per protein), It is

interesting that the ideal wei@;.'1t distribution calculated for the hypothetJ.cal macromolecule (figure 9) gives values of six, seven or eight depending on the m. w. of the protein. Considering the

variety of animals and shapes containing chitin, one may suspect some universality of these molecular weights a'1d their associated @ligosaccharide lengths.

-I1 I

' I I l l DISCUSSION OF RESULTS l PARTE j Protein Content

The deterndnation of the protein content by difference raises I a question of accuracy. When the lipidl.s, moisture and inorganic ! material have been removed, the remaining weight should be completely accounted for by the protein and carbohydrate contents . · The amino

acid analysis results indicate that almost all of the protein is

removed by KOH treatment with only minor persisting amounts of amino

acids, principally lysine. But is the carbohydrate degraded at the

same time? Testing the supernata~t in the KOH treatment periodically,

dces not reveal any c:harable m;o~.terial. Unfortunately this does not

resolve the question since the carbohydrate might be degraded in base

to compounds which may not be detected by charring. The stability

of thto carbohydrate polymer to base (6N KOH at 100°C) was tested,

and showed no·appreciable loss in three days. But the issue is not

settled. The surviving carbohydrate does set an upper limit of 45%

on the protein and the surviving protein in the amino acid analysis

sets a lower limit of 31%.

Since there was appreciable loss during the acid hydl•olysis,

especially of tryptophan and methionine, neither of which were found

at the end of a 16 hr hydrolysis, the higher value is more attractive.

The direct results might be even higher if any appreciable jnorganic

residue remained in the test sample or if the humidity in the testing I 65 lI laboratory required a higher moisture factor than was applied. The I 1 j results could be inproved by determining the tryptophan content in a

separate determination by basic hydrolysis. Methionine, if present,

can be determined by using a shorter acid hydrolysis time.

When the protein and carbohydrate found after byc;trolysis were ! compared, the results agreed within 1% for the two sarr:ples even though l a subtle error was possible. The carbohydrate content (and hence the

1 distribution) of.sample #1 was evaluated with a standard which was 1 made simultaneously with sample #2 but with a different ninhydrin solution. The agreement suggests a negligible erTOr in this case, \ but a second consideration is more significant. The protein content

1 was determined with the aid of amino acid standards which were not i hydl"'lyzed for 16 h.r. '!.he carbohydrate standard received the same tr·eatment·a.s the samples, so that the hydrolysis loss was compensated

l in for the calculation. This means that the protein found is low 1 J and that the 31/69% distribution should be corrected toward the I l 45/55% value. The chromatograms can be re-evaluated when graphs of I amino acid ilydrolysis are available. Applying a reasonable value of I' I 10% hydrolysis loss to the protein gives a 43157% distribution. i Thus a large amount of protein is found in the Velella float and is not all removed by extensive washing. Is this protein a com­

ponent of an adjacent membrane? Is it perhaps some component of the

mesoglea which becomes enmeshed in the carbohydrate matrix? Or is

it a true component of a glycoprotein macromolecule of which the float

is composed? 66

The Millon and ninhydrin treatments indicate that the protein is evenly

distributed in the lateral dimensions of the float. It was possible

to delamjnate the formalin preserved floats, and thls suggests that

the initial synthetic process is the development of a membrane-sheet

\~lich is of uniform thickness and topolo~cally continuous over the

entire outer surface (figure 10). The detail of the horizontal com­ ponent (figure 4) indicates that this membrane sheet is completed 1 before it contacts the prior layer. The picture emerges as an al-

J ternating mitosis-synthesis cycle, possibly circadian. The growth l process is reminiscent of a man constructing a ladder as he climbs. This model is supported somewhat by the observation that continuous 1 synthesis is illogical in view of the structure and that mi:tosis I and synthesis generany are considered mutually exelusive cellular l .events. The failure to find a membrane between the float and the

adjacent cell layer of the preserved specimen seems to elirninate li thls CtS a possible site for the protein. The protein is removed by 1 treatment vdth KOH without disturbing the shape. The filtrate does l not contain charable material. Examination of the formalJ11 treated floats during KOH treatment reveals large patches of material sloughing off the float. These events seem contradictory. If the "patch"

material observed sloughing off is pure protein, how is the internal

protein removed? The patches were not analyzed and perhaps the observed

darkening and shrinking of tl1e formalin treated floats result from

incomplete removal of protein. If the "patch" material is a portion ] 1 of the outermost layer, which has been deg;raded by formaldehyde, it 67

should contain protein and· carbohydrate, an hypothesis not investigated. . ljQ 45 . Formic acid is reported to "dissolve" chitinious material ' and

perhaps this is a related phenomenon. Formic acid and formaldehyde both react with proteins. A study which combined these reagents with the demonstrated acid and base effects might prove effective in deter- mining the exact organization of the layers.

The amino acid analyzer results do not reveal the number of proteins which are present, whether there are disulfide links (the

presence of half-cystine indicates the possibility), or other protein­ j protein crosslinks, or 11hether the true molecular weight is a multi- l l ple of the minimum calculated. The similarity between the samples ,\ of whole float material gives ..;onfidence in the results. ~lhen the l vertical and horizontal components were analyzed separately a differ- ~ l ence was obtained which has not been studied further. Initially this j seems to indicate at least two d:H'ferent proteins in the float, one 1 for the vertical layers and another for the horizontal layers . The .I I l layers are observed to be smoothly continous at the junction and such a conclusion seems untenable. Sequencing the protein ( s) may

resolve the question.

The presence of slx lysines in the mininnllll m. w. protein and its

persistence in the KOH treated float material suggested that a special

relationship may exist between lysine and the carbohydrate. Lysine was then given special attention. X-ray diagrams of "chitin" from other animals19 indicate a recurring structure of' six and eight

monomer distances. Electron micrographs of "chitin" from hymenopteran 68

41 ovipositor indicate a siX-sided carbohydrate ,structure surrounded by

protein. While not confirmed, the conversion of the six monomer pattern lnto the eigj:lt m:momer pattern when the protein is removed

suggests that the protein has six linkages per unit. The amounts of glutamic acid (7-8 residues),.proline (5-6) and valine (5) make them I candidates for such linkages, and the linkages may involve more than l one amino acid. If a one proteln model w:),th six links to an octasacc­ 1 har.ide is proposed so that all of the evidence from the literature ' 'I and this analysis is satisfied, a protein with 44-60 amino acids I (m.w. 7,000) results (figure 9). The name "velatin" is suggested for this protein(s) in order to emphasize species specificity.

What can be said about the relationship of the protein to the lI !' moisture, the lipid and inorganic contents? 'l'he relative moisture content of the l\OH treated materia}. (all carbohydrate) is the same II _j as 1n the whole float so that the protein seems to be as hygroscopic l j as the carbohydrate. The presence of one water molecule per amino

1 acid seems to be lndicated for the protein in the intact layer at room hwnidity. The lipid (or ether soluble component) defies evaluation other than its possible participation in the organization of the f1oat at

some level. It might participate in a hydrophobic-hydrophilic trans- ition associated with the aggregation phenomena. The reports, of microtubule rear,gregaUon3•54 describe a calciwn

dependence. The presence of calciwn in the whole float and its presence

after KOH treatment make possible its participation in the aggregation II I l j events~ The presence of a calcium-specific protein in other jelly­ fish implies that such a protein is to be expected in Velella, but

there is no indication that aequorin is identical to "velatin" or·

i that velatin is calcium specific. j ' What significance can be placed on the amino acid/carbohydrate

monomer correspondence? The results indicate that spatially the I carbohydrate and prote.in are equivalent. How can we remove such a I volume of protein without destroying the structure? Do the carbohydrates i rearrange to fill the void? The lateral shrinkage· is minor, so any significant rearrangement 'must result only in a thinner layer and would

not be observable. One gets the impression that the protein is not I ~ very important in the ll'.aintenance of the shape, although chitinase I 'l I and cW.tobiase do not degrade the structut'e whHe the protei.YJ is ' ~ 11 22 _j present • . (Collagen is protected from collagenase by a glyco­ protein in a sirrrUar vTay36 ,52). _ Does the -protein exercise -its bio'-'

1 logical function and specificity in .the initial construction of the j layer? The overall shape of the float seems to be determined by

l differential mitosis in the adjacent cell layer, the layer itself

l being sufficiently flexible (at least initially!) to match the contours

of the previous layers. The prote:in funct:Lon seems limited then to

the or'ganization of the layer and the layer-layer interactions. It

is spatially oriented relative to the carbohydrate chains so that

the glycosidic linkages are shielded from marauding enzymes.

The reaggregation of the centrifuged wash:ings might tell more -1 and the significance of such a study is ind:l.cated by the protejn-

l1 70 I

I carbohydrate participation jn specific cell-cell aggregation and the l . failure to reaggregate after treatment with trypsin29 ,3l,47 ,55. l To what extent does velatin participate in the extracellular l building of the layer? Since cellulose (a carbohydrate) and collagens· l (proteins} both aggregate spontaneously no preference is apparent. l . There is probably a concerted effect. ! :1 i CHAPTER 5

CONCLUSION

I How does one evaluate scientific work? For analytical results l and personal observations the author's statement of reproducibility invites ready confirmation if the reports are sufficiently detailed.

Another evaluation requires an objective appraisal of how the investiga-

tor's time and sacrifice have been rewarded. What really has been

1 accorr~lished? What value is there to the average person of a study 1 of "jellyfish bones"? The words themselves invarlably provoke humor and ridicule. The objectivity of .the author's evaluation is thus 1 l confronted by his desire to find significance in the results and the disqomfortin?; suspicion that nothing which could not have been pre- l1 --- ~ i d:icted was found. A reasonable middle ]Jath is perhaps a statement i ! of what has been confirmed, what is felt to be new, and what possi- 'I 1 bilities are suggested for further work.

The presence of chitin and protein in Velella float was con­ firmed and the common value of 45/55% for the weight distribution was found. Calcium was again found associated with connective

tissue. The carbohydrate was shown to be a iJ -liYlked polymer of N-

acetylglucosamine, and the spontaneous aggregation of chitinous

material was again observed. 1 I The definition by Hunt 9 of chitin as a protein-carbohydrate I complex conveys accurately the general picture found in Velella, I although some indication of the N-acetylglucosaminyl oligosaccharides 72

and the associated speci.es-specific(?) protein as a glycoprotein_

I subunit in sequentially synthesized layers will probably prove to be a more descriptive identification. When all of the levels of

organization and their linkages have been determined the definition

will cause less difficulty.

Some hesitation attaches to the designation of a finding as

"new" and perhaps a )Jl)re tentative phraseology would be more appro-

priate. However, it is cautiously put forward that the statement

by BrimacoiPbe and Webber that "Precise methods for the quantitative

deterrrdnation of chitL~ do not exist because the isolation and puri-

fication of the polysaccharide normally result in its modification. 4 1 Moreover, it is difficult to assess the purity ·of chitin " should J I be modified for Velella float material. The basic treatment of chitin ~. at room temperature was shown to give a p:mduct of defined purity,

-l·;; . anu the eleinerital analysis Seefils to-reselve the question of the degree .. 1 I of N-acetyJ.ation. The optimum .hydrolysis conditions for chitin uere

found and the application of Randle and Morgan's colorirretric_ deter­

mination methods39 were shown to be useful for chitin studies. The

absence of phosphorus was noted. The utility of the amino acid analyzer

was demonstrated for chitin investigations· and as an analytical tool

in assaying purification and separation techniques. An amino acid

analysis was obtained which was reproducible and quantitative deter-

minations were fo1md feasible for both the protein and carbohydrate.

The calculations of the amino acid results provided the basis for the construction of an hypothetical glycoprotein which seems to correlate 73

much of the evidence in the chitin literature. The author hopes that

a good case has been made for reporting amino acid analysis results

in mole ratios rather than the more common "residues per 1,000 residues".

The observation that the layers can be delaminated with formalin treatment should prove useful in the future and suggests a sequence

of events in the .morphological development of the float. The ob- servation of a topologically continuous concentric membrane-like (TCCM) structure provided an unexpected dividend for the author as it 58 provides support for a general hypothesis of biological activity which is based on the TCCM concept.

The Velella investigation seems to offer new insight into a number

of biological events. The gap between DNA and final shape seems .

somewhat narrower in the case of Velell.9;_ and hope.fully the ir1~ennediates can be j_solated and identified. The relationsr..ip between specific cell- cell attachment, differential mitosis, and the extl'acell\J.llar matrn: can probably be determined more readily in Velella than in a more

corq;Jlex animal. The expected phylogenetic progression of structural proteins can be studied. using velatin as a basis . A corq;Jarison of

the sequence of velatin with other structural proteins is to be eagerly anticipated. New insight is provided. into the general synthesis of

membranes, in which. glycoproteins and glycolipids unite into a continuous sheet, and into mucopolysaccharide "mucous" layers, in .which they do not . The peculiar correspondence in the amino acid and carbohydrate mole ratios suggests an undiscover·ed relationship between proteins

and hexose-hexosantne, and an explanation for the use of these par-

----;!1 ticular molecules for morphological events. 74

The work which is left to do is humiliating on the one hand and

in.c;pirational on the other in the offering of a lifetime of challenging

pursuits. As a reminder for further work one might mention the various linkages awaiting study, the amino acid sequence of velatin, the oligosaccharide length, the isolation of the glycoprotein fron1 the

mesoglea, the position of the oligosaccharide linkages (presumably

1,4), x-ray diffraction comparisons with other chitinous materials, detergent separation methods, a study of the interrelationship of light-

sensitive calcium-specific proteins and of purple pigments in circadian rhythms, the relationship of calcium, silicon and water to the matrix, the relationship between calcium and the contractility of the muscular margin, and the medical uses of a chemically defined chitin (intra­

articular injection of chitin induces an arthritic condition33). This list is incomplete but it is clear that Velella. represents a scien­ tific feast for many disciplines.

An extension of the introductm•y quotation might be appropriate

for a final statement. The extracellular matrix has its beginnings within the cell, and there is a continuum which jncludes the goJ.gi apparatus and the cell membrane surface37 . The function of the matrix

is concerned w.ith all substances, structures and events which occur in the extracellular space: e.g. membranes, tissue fluid, blood, ion concentrations, phagocytosis, imnune response, hormones, toxicity, synaptic phenomena, cell-cell interactions, nutrients, and gas ex- change. 'fue "complete knowledge of the structure and function of the matrix" is thus an ambitious goal. Hopefully the present work is 75

L a contribution, J One is naturally reluctant to conclude an obviously incomplete work; the author therefore announces •...

-a beginning-

1 l 'i I l _J ~ ~ .

J I

1 REFERENCES

~ 1 1 I I

1 -lj l ' 77

REFERENCES

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1 2. ·Barker, S.A., A.B. Foster, M. Stacei and J.M. Webber (1957) Chern. Ind. (London) 208; and (1958) J. Chern. Soc. (1958) 2218.

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11.1. Franzblau, C., B. Faris, R.W. Lent, L.L. Salcedo, B. Smith, R. Jaffe, and G. Crombie (1970) "Chemistry and Biosynthesis of Crosslinks in Elastin" in Chern. and Mol. Biol. of the Intercellular Matrix, E. Balaz ed., Academic Press-;-New York, val. 1, pp. 617-639. See also Yannas, I.V. (1972) Rev. Macromol. Chern. 8, 49; and W. Traub and K.A. Piez (1971) Adv. Protein Chern. 2b, 243 which gives a comprehensive survey of the various-pro­ posals for crosslinking in collagen. -- 1 I 78

I Also valuable ia J .G. Nold, A.H. Kang and J. Gross (1970) Science 170, 1096 which presents "Conclusive evldence that each TC \tropocollagen) molecule in mamallian skin is made I up of tw.o alpha 1 and one alpha 2 chains •... (there is) agree­ ment that the alpha chains are crosslinked intramolecularly by bonds located in their respective non-helical N-terminal -ends -and-formed by -a;Ldol Gon

15. Green, H. and B. Goldberg (1963) Nature 200, 1097; and (1964) J. Cell Biol. 22, 227. - - See also H. Green, B. Goldberg and G.J. Todaro (1963) Exptl. Cell_Res. 31_, 441.

16. Gross, J., B. Dumsha and N. Glazer (1958) Biochim. Biophys. Acta 30, 293.

17. Hackman, R.H. (1955a) Aust. J. Biol. Sci.§_, 83; (1955b) Aust. J. Biol. Sci. §_, 530. See also J. Gross and D. Kirk (1958) J. Biol. Chern. 233, 355; J. Gross (1964) Science 143, 960; and J.M. Cassel -(19~70)-National Bureau of Standards-Report 10271,- u.s. Department of Commerce.

18. (1960) Aust. J. Biol. Sci. 13, 568; (1962) Aust. J. Biol. ScL _15, 526.

19. Hunt, S. ( 1970) Polysaccharide.:.Protein Complexes in Invertebrates, Academic Press, New York, chap. 8. - -

20. Hyman, Libbie H. (1940) The Invertebrates, McGraw-Hill, New York, vol. 1, pp. 90, 300-301, 475-485.

21. Jackson, D.S. and J.P. Bentley (1968) "Biology of Collagen",­ part A, in 'I'reatise o~ CollageQ, B. Gould ed., Academic Press, New York, vol. 2, p. 1B9.

22. Je&~aux, C. (1963) phitine et Chitinolyse, Masson, Paris.

23. Kaestner, A. (19611) Invertebrate Zoology, trans. from the 2nd German edition by H. and L. Levi, Interscience Publishers, New York, vol. 1, pp. 43-88.

24. Katzman, R.L. and R.lv. Jeanloz (1970) "The Cru:bohydrate Chemistry of Invertebrate Connective Tissue" in Chem. and Mol. Biol. of the Intercellular Matrix, E. Balaz ecr.-; AcadeiTLi.c Press, New York, vol. 1, p;- 217. - 79

The authors state that "GlucosylgalactosyThyclroxylysine is apparently widely distributed throug.I-Jout the animal king­ dom as the carbohydrate moiety of collagen. Thus far, no other carbohydrate moiety has been found."

25. Kent, P.W. (1964) in Comparative Biochemistry, M. Florkin and H.S. Mason eds.,'Academic Press, New York, vol. 7, p. 93. See also P.W. Kent and M.W. lfuitehorse (1955) Biochemisj;£y_ _ of th~ Arninosugars, Academic Press, New York.

26. Kalthoff, I.M. and E.B. Sandell (1952) Textbook of Qua~titative Analysis, 3rd ed., Macmillan, New York, p. 339.

27. Meyer, K. (1970) in Chern. and Mol. Biol. of the Intercellular _ Matrlx, E. Balazect:", Academic Press,New York, vol.l, pp. 5-24.

28. Miller, E.J., G.R. Martin and K.A. Piez (1964) Biochern. Biophys. Res. Cornrn. ~7, 248; and E.J. Miller (1971) Biochern. Biophys. Res. Cornrn. _2, 444.

29. IVIoscona, A.A. (1971) "Embryonic and Neoplastic Cell Surfaces: __ AYailability _of Receptors for Concanavalin--A and w'heat- -Germ Agglutinin" Science 171, 905; and .(1968) in In Vitro, M.M. Sigel ed., Williams ancf Wilkins, Baltimore, vol-;- 3, pp. 13-21.

30. Neville, A.C. (1965) Q.J. Micros. Sci. 106, 315.

31. Nicolson, G.L. and R. Yana.gimachi (19'{2) "Terminal Saccharides on Sperm Plasma Membranes: -Ideritificatiori by Specific Ag-' glutlnins" Science 177, 276.

32. Nissan, A.H., G.K. Hunger and S,S. Sternstein (1965) "Cellulose" . in ~Sy; of Polymer Sci. and Tech., Wiley; New York, vol. 3, p. 1 .

33. Page Thomas, D.P. (1970) "Experimental Arthritis and Acid Hydro­ lases" ln Chern. and Mol. Biol. of the Intercellular Matrlx, E. Balaz ed., Academic Press, New York, vol. 3, p. 1'7~

34. Partridge, S.M., D.F. Elsden, J. Thomas, A. Dorflnan, A. Telser, and P-L. Ho (1966) Nature-209, 399.

35. Pearson, F.G., R.H. Marchessault and C.Y. Liang (1960) J. Polymer Scl. 43, 101; and (1960) Biochirn. Biophys. Acta 45, 499.

36. QmntarelH, G. and M.C. Dellovo (1970) in Chern. and Mol. Biol. of the Intercellular Matrix , E. Balaz, ed. , Academic Press, I New York, vo1. 3, p. 1403. -1 ~'he authors state that "The results reported here sustain I 80

(our earlier opinion; 1966, 1967) and are in agreement with the findings of Steven et al (1968), that a glycoprotein linked with collagen prevents in some as yet unknown manner, the hydrolysis of the collagen fibers by collagenase. 11 See also G. Quintarelli and M.C. Dellovo (1966) Histochernie 7, 141; and (1967) Histochernie Q., 216. · -

37. Revel, J.P. and S. Ito (1967) in The ~pecificity of the Cell Surface, l~arren and David eds. , Prentice-Hall, Englewood Cliffs, New Jersye, p. 2ll. ~ 38. (1970) "Role of the Golgi Apparatus in the Elabor- l ation of Matrix Glycosarninoglycans" in Chern. and Mol. Biol. ~ of the Intercellu:lar Matrix, E. Balaz e~Acadernic-PresS, j New York, vol. 3, p. 1485. ,, 1 39. Rondle, C.J.M. and W.T.J. Morgan (1954) Biochem. J. 61, 586.

40. Rudall, K.M. (1955) "The Distribution of Collagen and Chitin" in Symposia Soc. Exptl. Biol £, 49. I 41. (1963) Adv. Insect Physiol. !, 257. l ~ 42. (1965a) Biochem. J. 95, 38P; and (l965b) in Struc­ ture and Funct~ on of ConnectiveTissue, Butterworth, London, i P• l91:-- - i• 43. Sandberg, L.B., T.N. Hackett and W.H. Carver (1969) Biochim. Biophys. Acta 18~, 201. 1 j 44. Schmitt, F.O., J. Gross and J .H. Highberger (1953) Proc. Natl. \ j Acad. Sci.(US) 39, 459. j 45. Schulze, P. and G. Kunike (1923) Biol. Zentr. 43, 556. I 46. Scott, R.P.W. and D.W. Grant (1964) Analyst 89, 179. I 47. Sharon, H. and H. Lis (1972) 11 Lectins: Cell-agglutinating and I · Sugar-specific Proteins" Science 177, 949.

48. Shimomura, 0., F. Johnson andY. Satga (1962) 11 Extraction, Puri­ fication and Properties of Aequorin from Aequorea" J. Cell Comp. Physiol. 59, 223-239. See also J.R. Binks, P.H. Mattingly, B.R. Jewell, and M. Van Leeuwen (1969) Fed. Proc. 28, 781; B. Kam:i.ner and J. Kimura (1972) Science 176, 4o6;-and C.C. Ashley and J. Ridgway (1970) J. Physiol-:-London 209, 105. -I 49. Smith, D.W., N. Weissman and W.H. Carver (1968) Biochem. Biophys. Res. Comm. 31, 309. 1 81

Spiro, R.G .. (1970) "Caxbohydl'ates in Collagen" in Chern. and Mol. · Biol. of the Intercellulax Matrix, E. Balaz ed., Adacemic Press,~ew York, vol. l, pp. 195-215. The author states: "The density of the (hydl'oxylysine­ linked) carbohydrate units varies from l per 1000 amino acid residues in scleral collagen to 27 per 1000 amino acid residues in lens capsule." It is further noted that in tendon collagen many single galactose residues are present with the gluco­ sylgalactosyL~ydroxylysine and galactosylhydroxylysine, while in the basement membranes almost all units are disaccharides and only glucosylgalactosylhydl'oxylysine is found.

Steiner, R.F. (1965) The Chemical Foundations of Molecular Biology, Van Nostrand, Princeton, New Jersey, p. 39b.

Steven, F.S., D.S. Jackson and K. Broady (1968) Biochim. Biophys. Acta 160, 435.

von Rippel, P. and T. Schleigh (1969) "Ion Effects on the Solution Structure of Biological Macromolecules" Accts. of Chern. Res. ?_, 257.

··Weisenberg;-R;C • · -e 1~727-"Microtubule -Formation- in--vitro in Solutions­ Containing Low Calci urn Concentrations" Science 177; 1104.

"Tei:ser, M.M. (1972) "Concanavalin A .:'\gglutination of Intestinal Cells from the Human Fetus" Sclence 177, 525.

Whaley, G. W. , M. Dauwalder and J. Kephart (1972) "Golgi Appaxatus: Influence on Cell Surfaces" Science 175, 596. Yannas, I.V. (1972) "Collagen and Gelatin in the Solid State" Rev. Macromol. Chern. ~' 49. Presented in a seminar at U. of the Pacific, Stockton, California, 12 July, 1972. In prepaxation. APPENDIX

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CD 1.0

Infrared Spectrum 4(a). Velella Float Material: (6N KOH, 8 days at 25°c) - '~··-''·~-~~~-~-~ '"-'=".. =·==L·==,

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\!) 0 Infrared Spectrum 4(b), Velella Float Material (.6N KOH, 8 days at 25°C) ! ~=~~~~-~~~

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FREQWENCY I CM ·1)

\0 ~ Infrared Spectrum 5(a), Velella Float Material (1N KOH, 24 hr at 100°C) ~~-'J""'".-~-~"""'-'· ~~~~~,-~-'.-~··<~~--~, ..... ~ ~~~~--'~,~~~-~-~-- ·····------·--~-~---'""'"1"'~'"'~-- -'~-~-=~--~~-~--~-~~

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FREQUENCY (CM-1) 1.0 1\) Infrared Spectrum 5(b), Velella Float Material (lN KOH, 24 hr at 100°C) --""~~-~ ...... ~- """"='-'-· ~~~~~ ~~~-~·~~-~~L .. _,,_,,.~.---'..'"""""""-'~d=-""''"''="'"----- ~·~-~~~~~~~

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F~EQUENC':' l CM ·1)

\!) w Infrared Spectrum 6(a), Crystalline Material From Acid Hydrolysate o:f:Velella Float Material i '-~~-=---·'"'"-"---'-·--'~·~·~·~-~- ""-~'--"--""~LJ==~"-'""~'"""'j_~~-=.w.~~~~~~- --.,-~·=~==~· ~··~~·~~-~~~~.

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\0 Infrared Spectrum 6{b), Crystalline Material From Acid -"=' Hydrolysate of Velella Float Material "~~~~"~~~---"'""""~~·

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FREQUENCY (CM·1)

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Infrared Spectrum ?(a). Velella Float Material (washed and dried) .. -- -~ -""~~~~""'~"-""''"-· -~-~-~---.~-~---,L~--~-.. -'~·-~~~ "'.- - ._,_ --~----~-' ~~-

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FREQUENCY {CM·')

'-0 0\ Infrared Spectrum ?(b), Velella Float Material (washed and dried) 97

Table 8

Systematic Identification Of Velella ~

Phylum Coelenterata (Cnidariar. Coelenterates. Symmetry radial or biradiall the individual either a cylindrical sessile polyp, often in colonies, or a free-floating bell-like medusa with much mesoglea; with stinging n~,;.atocysts; digestive cavity (enteron) sac-like, sometime'S branched; soft t·entacles about mouth; no anus, no head, no other organ systemsJ nervous system diffuse; some with eyespots, or statocystsj. r·eproduction i usually asexual in polyps ·and sexual in medu.~_~e;_ -l dioecious or monoecious; no sex ducts1 all aquatic, '! chiefly marine, attached. or floating; Lower Cambrian i ' to Recent, 10,000 species. Class Hydrozoa, Hydroids (and some medusae). No stomodeum; !j j I enteron lacks partitions and nematocystSJ m~sog,lea. ' noncellular! medusae usually small and with velum (oraspedote) 1 chiefly in shallow salt vmters1 colonial or solitary, 3,700 species, Order Hydroida, Polyp generation well developed, solitary or colonial, usually budding off small free medusae that bear ocelli and ectodermal statocysts. Suborder Chondrophora (Physophorida). Polymorphic, colonial, free-floating! gonophores feed as medusae, upper end

of colony a~loat or pneumatophore, Yelell~, float thin, with erect sailJ Porpita, float disc-shaped.

(from Storer, Tracy 1. and Usinger, Robert L•• 11 General

Zoology," 4th ed. 1 McGraw-Hill, New York, 1957) 98 Table 9

Phyla Of The Animal Kingdom

Protozoa Mesozoa Porifera Coelenterata Ctenophora Platyhelminthes Nemertinea Entoprocta Aschelminthes Acanthocephala Ectoprocta Phoronidea Brachiopoda Mollusca Annelida Sipunculoidea Echiuroidea Arthropoda Chaetognatha Echinodermata Pogonophora Hemichordata Chordata 99

(Jl '"CJ ro ·.-l I () ..::}- 0 ..::}- '"CJ ~ "'l • >< " "' (/) I ~- ',j s:: (]) t\0 ..-J ~ s:: (l) •rl ~ +> ?n s::: ~ 0 •.-l ·.-l ~ li< ro P< +>s:: ~~ 0 0 0 s:: 0 ro ...... s:: 0 (l) s ro 0 (l) 1-1 ,:... () P< ro (l) :2> ~ ...... 0 !\1 •.-l 0 +> •.-l ro +> s 0 0 [/) ~ ~ 100

(a) two dimensional representation (end view)

(b) three dimensional· schematic representation illustrating TCCM model

Figure 10, Hypothetical Construc.tion of Velella la.i§; Float Based on a 'l.'opologically Continuous Concentric Membrane (TCCM) Model