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The Chemical Analysis of Velella Lata Float

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The Chemical Analysis of Velella Lata Float 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 Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Biochemistry, Biophysics, and Structural Biology Commons 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.
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