South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange
Electronic Theses and Dissertations
1972
Biosynthesis of Canavanine
Shu-Chiung W. Chen
Follow this and additional works at: https://openprairie.sdstate.edu/etd
Recommended Citation Chen, Shu-Chiung W., "Biosynthesis of Canavanine" (1972). Electronic Theses and Dissertations. 4639. https://openprairie.sdstate.edu/etd/4639
This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. BIOSYNTHESIS OF CANAVANINE
BY
SHU-CHIUNG W • CHEN
A thesis submitted in partial fulfillment of the requirements for the degree Moster of Science, Maior in
Chemistry, South Dakota · State University
1972
SOUTH DAKOTA STATE UN·IVERSITY l ARY BIOSYNTHESIS OF CANAVANINE
This thesis is approved as a creditable and independent investigation ·
by a candidate for the degree, Master of Science, and is acceptable as · meeting the thesis requirements for this degree. Acceptance of this thesis does not imply that the conclusions reached_ by the candidate are neces sarily the conclusions of the maior department.·
ryisff'Adviser Date
Head, Chemistry Department Date ACKNOWLEDGEMENTS
The author wishes to express her sincere appredation to ·
Dr. Terry J. Gilbertson under whose direction and advice the research was carried out. A word of thanks also goes to the Chemistry Department .and
Pharmacy College for supplying the materials and equipment to make thfa work possible.
The duthor also wishes to extend her gratitude to Mr. Jeong-Shwu Liu for drawing the figures. TABLE. OF CONTENTS
INTRODUCTION Page
Discovery of Canavanine ......
Physical Properties and Structure Proof • • • • • I• • • 1
Methods of Isolation ...... 5
• • • • . . Biosynthe sis ...... 9
PROPOSAL ON BIOSYNTHESIS OF FOUR CARBON CHA IN OF CANAVANINE
Methionine Hypothesis • • • • • • • • • • • 10
Glutamic Acid Hypothesis • 91 • ...... 12
RESULTS ...... 16
• ...... CONCLUSION . . . . 19
EXPERIMENTAL
. . Description of Instrumentation Used • � • I • • • • • • • • • 21
Description of Pentacyanoammonioferrate
and Ni nhydrin Tes ts . • • • • • • . . . . • • • 22
Paper and Thin-layer Chromatography Used • • . . • 25
Isolation of Canavanine • • • ...... 28
Degradation of Canavanine • ...... • . . 32
Method of Feeding Plants and Growth of Plants ...... 34
• • • • • • • . . . Literature Cited • • • . . . . • 35 TABLE OF FIGURES
Figure Page
1 • Reactions of canal ine ...... • • • ...... • • 3
2. Van Slyke's reaction for canavanine and
Enyzmati c degradation of canavanine ...... • ...... 4
3. Synthesis of Canavanine from canaline- • • • • • • • • • • • • • • 6
4. Reaction of 0-ethers of hydroxylamine with ha lide acid 0 • . . • • 7
5. Reaction of canavanine with hydrobromide • • • • . . • • 7
6. Synthesis of canavanine from Y-butyrolactone • ...... 11
7. lntramolecular nucleophilic displacement of
. S-adenosyl methionine • • • • • • • • ...... 12
8. Proposed biosynthetic route from methionine • • ...... 13
9. Proposed biosynthetic route from glutamic acid • • ...... • • 15
10. Activity of effluent from AG-50W-8X column • ...... 18
11. A standard curve of PCAF test for canavanine sulfate • ...... 24
12. A ninhydrin test and a PCAF test for effluent from
AG-50W-8X column • • • • • • • • • • • • • • • • • • • · • 30
13 0 A ninhydrin test and a PCAF test for effluent from
IR- 4B co I umn • • • • • • • • • • • • • • • . . . . . • 31
14. Degradation of canavanine • • • • • • • • • • • • • • • • • • • 33 1
INTRODUCTION
DISCOVERY OF CANAVANINE
Canavanine, o{ -amino- Y-guanidoxy-butyric acid, was first discovered in
Jack bean {Canavalia ensiformis) by Kitagawa and Tomiyama (1-3). The com- pound is basic and is found free in the non-protein fraction of Jack bean. It is soluble in 50% alcohol, is precipitated as a viscous mass in absolute alcohol, and can be hydrolyzed by the enzyme, arginase, to urea and a new diamino acid, canaline, C H o N • Subsequently in 1939 Damodaran and Narayanan 4 10 3 2 found it in the seed of C.Obtusifolia. (4) So far it has been found mainly in the seeds of Leguminosae, subfamily Papilionoideae. The concentration is occasionally as high as _3.5% of the dry weight. (5-8)
PHYSICAL PROPERTIES AND STRUCTUR E PROOF
Canavanine H2NC(NH)NHOCH 2CH 2CH(N�)C02H (9) _ ° 2 mol.wt. 176.18 mp 184 (o<) g = +7.9 (w •. c.=2)
It crystallizes from alcohol but crystalline form is not reported. It dissolves in water but not in alcohol, ether or benzene. It has some derivatives as listed below:
Sulfate: crystallized from alcohol 0 2 mp 17 deco mp. (o< ) I� = + 19. 4 1 0 Copper salt: mp 205-8 decomp. 0 Tribenzoyl: mp 86 decomp. 2
° 163-4 Picrate: mp ° 212 Flavianate: yellow needles from water mp
o The constitution of canavanine C 5H12 3N4 has chiefly been determined H o N , by studying the simpler amino acid, canaline C 4 10 3 2 which is formed
together with urea by the degradation of canavanine with the enzyme, arginase. to Kitagawa assigned the structure, H2N-O-C� 2CH 2CH(NH2)C02H, canaline
based on the fact that it contains N which is not detected either by Van Slyke's
1). H reaction nor by the formol reaction (Fig. On reduction with 2 and Pt 1 1 black in AcOH or MeOH, it absorbed mole of H2 and set free mole of
N H3 • A substance which was regarded as identical with synthetic o( -amino 1). Y-hydroxybutyric acid was isolated from the reaction (Fig. No free hy
droxyl group could be found in canaline. When canaline was warmed with
mineral acid, it did not give a lactone. The hydroxyl group appeared in the
N Y-position after catalytic reduction. Therefore, this non-amino group on -ON� (10-1.3). canaline was considered to be combined with 0 as 2
Canaline also was synthesized from ethyl o(-benzoylamino-l"-iodobutyrate.
(13, 14) The natural and synthetic products were shown to be the same, namely, o<-amino-Y-0-hydroxylaminobutyric acid. 7. 93), From the properties of canavanine, its basic character (pl its fission into urea and an amino compound, and the reaction of only two of its
four nitrogen atoms with nitrous acid in the Van Slyke determination of amino .. nitrogen, (Fig .2) Kitagawa et aL suggested that the guanidine group 3
Van Slyke's Reaction
Forrnol Reaction
HC-HII > 0
HN-CH20H 9. N-(CH2 OH)2 HC-H H2N-0-(CH2kCHtr-OH > H2N -0-(CH2)2-CH�-OH 0 0
Reduction with H2
1. ·Fig. Reactions of canaline. 4
+ HONO
· O OH
2. Fig. Van Slyke's reaction for canavanine.
arginase
2. Fig. Enzymatic degradation of canavanine. 5
H2N·C( :NH}•NH was probably present in canavanirie. They prepared
dibenzoyl canaline from canaline with BzCl and NaOH. When it was
10% o( treated with Ac20 first, then H2S041 -benzoyl canaline was ob
tained. o<-benzoyl canaline reacted with methylisourea and MeOH in the 10% cold. The precipitate was decomposed with HC1 then �lavianic acid 3) was added. Canavanine flavianate was obtaJned. (Fig. This confirmed (15) that canavanine is a guanido derivative of canaline.
Then 0-ethers of hydroxylamine reacted with hot halide acids, they
yielded the alkyl halide and hydroxylamine. (Fig. 4 ) Gulland and Morris (16) treated canavanine with concentrated hydrobromic acid in a sealed ° 5 160 . tube for hours at Canavanine was converted into ammonia,
guanidine, and a substance which must be regarded as o(-amino-¥-butyro 5) lactone hydrobromide in view of its properties. (Fig. When this sub
stance was heated with concentrated hydrobromic acid, it yielded opti•
cal ly inactive Y-bromo-o<-amino butyric acid hydrobromide, i den tical ·
o< with a specimen prepared by the same �ethod from synthetic -amino-
Y-butyro-lactone. These results proved canavanine has the structure,
METHODS OF ISOLATION (2) The Jack bean plant is still the main source of canavanine. Kitagawa
50% extracted canavanine from Jack bean with alcohol and treated it with
flavianic acid but the %N was lower t�an the ·theoretical value··for this 6 .
CsH5COCI
Ac20 cool >
3. from Fig. Synthesis of canavanine canaline 7
- R-O NH2 + HCI > RCI + NH20H
4. Fig . Reaction of 0-ethers of hydroxylamine with halide acid .
�H2·HBr �H2·HBr CH2CH-C-OH CH2CH2CHC=O + HBr ----.) Br-CH2 I O_J. 0
Fig. 5. Reaction of canavanine with hydrobromide. 8 flavianate. In 1937 Kitagawa and Tsukamoto {17) recommended the destruction of the impurity by digesting the first crude canavanine precipitate with 10% HC1. They reported that this gave a high purity canavanine flavian ote1 but they did not give a detailed method nor the yield. In 1935 GuHand and Morris (16), who also had the same difficulty, suggested the purification of the base liberated from the flavianic acid �a lt by conversion into the rufianate. In 1939 Damodaran {18) treated the crude canavanine with a solu- tion of basic lead acetate, and then with flavianic acid. He got a. high purity flavianate successfully, but had trouble removing the excess lead. In
1962 a method was devised using ion-exchange resin to prevent the formation of desaminocanavanine with cold 99% alcohol from the concentrated canavanine extract, then treated it with flavianic acid, and decomposed the flavianate with hot saturated Ba(OH) • The filtrate was passed through IR-48 2 - (OH form) ion-exchange resin to remove the impurity. The effluent was con- centrated under reduced pressure and canavanine crystallized upon the addi- tion of absolute alcohol. The merits of this method are that it gives the free canavanine in high purity, easily and in a good yield, directly from its flavianate. (19)
In the experiment of the author, the filtrate from decomposition with
Ba(OH) was passed through AG-50W-8X resin and ca navanine•2HC1 was 2 eluted with 4N HCl. The effluent contain_ing canavanfoe.2HC1 was passed through IR-4B (OH-form) resin to get free canavanine. · 9·
BIOSYNTHESIS
Several authors have (7, 20, 21) reported that canavanine was syn-
thesized in the pod and transported to the maturing seed. In 1970,
Gerald A. Rosenthal (22) studied the ontogenesis of canavanine formation
and confirmed this assertion. It has been implied that canavanine acts as a
nitrogen source in coniunction with arginase 5md urease for the developing
embryo. (23-24) 14 In 1961 Tschiersch (25) iniected glycine-1- c into the immature - fruit of Colutea arboresceus (Bladder Senna) and labeled amino acids were
found in the soluble amino acids and in protein hydrolyzates of both pod
and seed after 12 and hours. The canavanine was labeled only slightly 48 14 after hours. He decided g ycine-1- c was not a specific precursor of 48 i canavanine. In 1969 Warren and Hunt (26) showed that circular disks cut
from surface sterilized slices of the green pod of C.Onavalia ensiformis would 14 fix and the activity of this carbon could be found .in isolated co2 cana
vanine. Canavanine was hydrolyzed with arginase and ratio· of the canaline the ·
to urea was al most 2 to 1 instead of the expected 4 to 1. They .suggested. that
a relatively inactive metabolic precursor pc::irticipated in canavanine bio . 14 synthesis. In 1970 Topfer, �t al. (27) reported �hat homoserine-4- c
was incorporated to a very small extent in the seed of Caragana s ponosa
(L.) DC. (Fabaceae}. So far the precursor of ca navanine is stil l unknown. 1 0
PROPOSAL ON BIOSYNTHESIS OF FOUR CARBON CHA IN OF CANAVANINE
ME THIONINE HYPOTHES IS
All the in vitro synthesis of canavanine have involved derivatives of
hydroxylamine. (14, 28) Nyberg and Christensen synthesized canaline by a
five step reaction scheme starting with Y-butyrolactone in 7% over-all
yield. Canavanine was prepared directly from canaline in 0.9% ·yield. c.
The synthetic.�cheme is illustrated in Figure 6. It seems likely that the biosyn
thesis alsoinvo lves a derivative of hydroxylamine.
Leete (29) proposed methionine as,a source of four carbon chains and
demonstrated that this was the case for th� biosynthesis of L-azetidine.-2- · . carboxylic acid as in Figure 7. The mechanism probably involves an intra
molecular nucleophilic displacement of S-adenosyl-methionine. The result
indicated that essentially all the activity of azetidine-2-carboxylic acid was
located on the carboxyl group. Therefore, we considered it possible that the
biosynthetic route to canavanine may have involved a nucleophilic displace
ment on an S-adenosylmethionine by a derivative of hydroxylamine.
Walker (30) has tried to find transamidinase activity in Jack bean.,' 1
Therefore, it may be possible . but no activity was detected. for the biosyn thetic route after canal ine formation to be similar to that for arginine. (31)
Walker has found that in many microorganisms the canavanine-fumarate
reaction occurs and Jack bean arginosuccinase can split the canavariino
succinic acid to canavanine and fumarate,. Figure 8 shows the propo�ed 11
K2C03
KCN HBr
ICH2CH2CH - CO - BrCH2CH29H-9 =0 I I Na! HN NH . HN NH �-i n-ac_e_t_o-ne-- / 'c/ , II cII 0 . o.
hydroxyurethane/.
a le. KOH
< HBr
0 . II NH20CH2CH2 fHC-OH NH2
6. F ig. Synthesis of canavanine from Y-butyrolactone. 12
adenosin CH2-CH2 I e I I
- CH3-S-CH2-CI H2 CH3-S-adenosine + H-N CHCOOH + f H2N - CHCOOH Fig. 7. lntramolecular nucleophilic displacement of S-adenosyi methionine.
synthetic route to canavanine if methionine is the precursor of the four carbon
chain. GLUTAMIC AC ID HYPOTHESIS
Hydroxylamine has been reported as a reduction product of nitrite in
green plants (32). Elliott has found that enzyme systems from bacteria and.
higher plants (33-39) cotalyse the reaction of hydroxylamine with gluta�ic
acid to form ¥-glutamyl hydroxamic acid. We considered it possible that
glutamic acid could be the precursor of the four carbon chain by a rear-
rangement and decarboxylation of Y-glutamyl hydroxamic acid. The
proposed biosynthetic route is shown in Figure 9.
Therefore, we propose to test these hypothesis by feeding methionine
1 4 4 -1- c and glutamic acid"'."1- 1 c. ff these amino acids are incorporated by
.the proposed biosynthetic routes they will label carbon one of canavanine. 13
. · I . Methionine . +. ATP > S-adenosyl methionine I adenosine 0
R-N-OH + H
. 0. . II R-N-0-CH CH -CHC-OH CH3-�-adenosine I . + H 2 2 NH2
+
0 0 0 II II . It .
H2NC-O-P-OH H2 N-O-CH2 CH2 CHC-OH -. ----�> a + I HO NH2
0II
I HO-P-OHI OH
Fig. 8 Proposed biosynthetic route from methionine.
2 6 9 t) 1 9 SOUTH DAKOTA STATE UNIVERSITY LIBRAR 14
0 0 0 0 II II ti . . 11. HO-C-CH2CHC-OH H2N-C-NH-O-CH2 C CH.C-OH I + H2 l -----..> NH2 NH2
+
0II 0II HO-C-CH2yHC-OH N 0 . II II H2N-C�N-O-CH2 CH�9Hc�oH H NH2
o NH 0 - II 0II HO-C-CH=CH-C-OH + 15
0II 0II
HO-C-CH2CH27H-C-OH + H2 NOH + ATP NH2
0 0 .· II II 3 - HO-N-C-CH2CH2CHC-OH + . ADP + PQ. H NH2
Q,, 0 0 0 " n · " H2N, " rearrangement . HO-N-C-CH2- CH2.CH C-OH HO-C-N-O-CH2CH2CHC NH2 OH · �
0II c --�> H2N-O-CH2CH2 yHC-OH + o2·.
NH2 \'\....-'
----�) rest of route the same as figure 8
Fig. 9. Proposed biosynthetic route from glutamic acid. 16
RESULTS
,., ' Methion:ine Feed Resu.f t ------· -- 14 - The methionine•1- c (o . l me/ml) was administered to the plants
for five days and the harvested fruit weighed 923 g. The yield of ° canavanine • 2HC 1 was 53 mg (0 .213 m mo les, mp 110-1 J.8 ) and the
radioactivity recovered was 10, 653 dpm .
Canavanine·2HC1 (27mg, 0. 108 m moles) was degraded with arginase
(25 mg, 24 units). The Dixanthydrylurea obta ined was 12 mg (0. 029 m -
moles, 26. 3%) in yield and was not radioactive. The radioactivity in the
canaline solution (3 ml) was 3107.4 dpm. The result of the degradatio.n
naline with ninhydrin was that no radioactivity appeared in of ca BoC03
(3 mg, 0.015 m moles 14. 3%) and the radioactivity in the ninhydrin
·mixture solution was 4787.2 dpm .
Glutamic Acid Feed Result ------14 The glutamic acid- 1- c {75 mc/m 1) was administered to the plant for
fourteen days and the harvested fruit weighed 17.6910 g. Owing to the
small amount of fresh pods, co ld conavanine• H SO (200 mg) was added to 2 4 . the ground Jack bean pods.
The radioactivity of each fraction from the AG-50W-8X co lumn was
counted using toluene scintillator. (Fig. 10) Most of the radioactivity was - 4N eluted with H o. Only a portion of the third peak (1 10-140 m 1 of 2 17
HC 1) showed a positive pentacyanoammonioferrate test. This portion was passed through an IR-4B column and eluted with distilled water. Thin-layer chromato- graphy using an Eastm�n 6060 silica gel p!ate showed the impurity of the effluent
I and the strip scanner showed that there was no radioactivity in canavanine, but radioactivity appeared in the area corresponding to arginine. All of the remaining effluent was applied to a thin-layer chromatography plate in order to ° separate canavanine. Free canavanine (2 mg. 180 decomp.) was obtained and was not radioactive. The radioactive area of the chromatogram (3000 dpm) was recrystallized with 100 mg of cold arginine . The yield of arginine was
92 mg and the radioactivity recovered was 2769.2 dpm (92 .3%).
Chromatography of the second radioactive peak (1 N HC 1 effluent,
3194.9 dpm} gave three spots (one yellow, one blue and one purple after spraying with ninhydrin) . The purple one fell on the radioactive peak as shown by the strip scanner. Cold glutamic acid· HCl {100 mg) was recrys-
tallized with the filtrate of the area corresponding to the purple spot and · the yield of glutamic acid was 91 mg. The radioactivity recovered was
2802.8 dpm (87.7%) .. !500
4eo ,, -· (Q .
.... 400 0 .
)> 0 350 ::!". < -· - ...... '< 0 300 ...... � � "'O �...... , c ..._,, (I) 250 :J - � ...., ... ·- a > 200 3 ... )> u G)I <( 01 150 0
I �CX> 100 x
8c 50 3:J .
0 20 40 60 20 40 60 80 100 10 30 50 70 90 110 130 150 170 190 210 200 . 10 30 50 10 30 50 70 90 110 20 40 60 80 100 120 140 160 180 � HiO. >k· 1 N HCI >� 4 N HCI •I - Eff I uent Volume ( m I) (X) 19
CONCLUS ION
According to the proposed biosynthetic route for canavanine from methion ine, all the radioactivity should be on carbon-1, but the result of the methionine feed demonstrated no activity on carbon-1, or the guanidoxy group, and all the activity on the remaining part. It did show that methionine was incorporated in the biosynthesis of canavanine, but was not a specific pre cursor of canavanine.
The result of the glutamic acid experiment indicated that glutamic acid was not the precursor of canavanine in the Jack bean. There may be several pathways that utilize the administered glutamic acid. One of these leads io arginine and the largest part of glutamic acid probably goes into the com pounds of the tricarboxyl ic acid cycle.
The two most frequent criticisms of any interpretation of negative bio synthetic evidence are : no evidence to show that the plant was making the metabolite during this stage of its life, and no evidence that the precursors reached the site of biosynthesis in the plant.
The evidence that the concentration of canovanine increases during the growthof the pod has been verified by several groups. (5-7) Since we fed the plants during the growth of the pods, the first criticism is not valid.
The second criticism is not so easy to answer. Since we only harvested the pods which were at a considerable distance from the site of administra- 20
tion of the precursor, we know that the precursor was transported to the pods
as the pods were radioactive. We can not be sure that they reached the· exact
· site of biosynthesis. Perhaps they did not. The only successful feeding experi
ments have been with sterile pod slices (26) so perhaps that must be tried be-.
fore vte can say that methfonine and glutamic acid are not precursors of
· canavanine. 21
EXPERIMENTAL
DESCRIPTION OF INSTRUMENTATION USED - Seed was ground with a Waring Blender first, then in a small General
Electric Mill .
Melting points of the compounds were taken on a Fisher-Johns melting point apparatus.
The pH values were taken with a Heath Servo Digital pH/volt Meter,
""°del EU-302A. TM The infrared spectra were obtained on a Beckman IR-33 Spectro photometer using KBr plates.
Chemical analysis of canavanine was performed by Galbraith
Laboratories Inc., Knoxville, Tennessee.
The optical densities were determined with a Beckman spectronic 20 spectrophotometer. The wave· length used was 570 mµfor the ninhydrin reaction and 520 m)Afor the PCAF reaction.
The determination of the radioactivity was done with a Model 3375
Packard Tri-Carb liquid scintillation counter. The discriminator settings used were 50-1000 units for the red channel and 50-100 units forthe green channel . The % gain used was 8.3 forboth channels. Two kinds of scin tillators, 2, 5-diphenyl o�·.azole, I, 4-bis-2-(4-methyl-5-phenyl oxazolyl) benzene, dioxane-naphthalene were used. The standard efficiency curve was prepgred by Terry Gilbertson . 22
The determination of the radioactive areas on thin-layer chromatograms was done with a Model 7201 Packard Radiochromatogram Scanner. The high voltage used was 1100 volts . The gas flow rate used was 200 cc/min. The scanning speed used was 2 cm/min. and the counting rate used was linear 2 range of 3x10 counts/min • .
DESCRIPTION OF PENTACYANOAMMONIOFERRATE AND
NINHYDRIN TESTS
Preparation of Pentacyanoammonioferrate (40)
Recrystallized sodium nitroprusside (10 g) was dissolved in 40 m 1 of concentrated NH40H and stored in the refrigerator over-night until all the nitrosoferricyanide had decomposed . This is shown when a few drops of the mixture no longer gives a red color when added to a solution of creatinine in lN NaOH (Weyl's test).
After 24 hours a mixture of greenish yellow pentacyanoammonioferrates
II and Ill separated out. The precipitate was filtered. The yellow penta- cyanoammonioferrate II was precipitated by the addition of absolute EtOH
0 ml ) to the filtrate . This precipitate was washed free of NH with abso (10 3 lute alcohol and dried and s1"9red in a vacuum desiccator over CaC1 2•
Preparation of Activated 1% Pentacyanoammonioferrate II Solution
Dry pentacyanoammonioferrate II (2 g) was dissolved in 200 m1 of I double distilled water. It was exposed to air and light for 24 hours and then 23 stored in a brown bottle in the dark.
Activated Pentacyanoammonioferrate Test {PCAF Test)
To 1 rTJ of canavariine soI ution was adCied 10 m of phosphate buffer I I {a mixture of equal • H volumes of 0.066M NaH2PO 4 20 and 0.066 M of Na HPO ; pH 7) and 0.5 ml of PCAF reagent. It was mixed well and 2 4 stood for 40 min. for color development. The optical density was read at
520 mµ . A standard curve for canavanine sulfate was shown in Figure 11.
Preparation of Reagents for the N inhydrin Test (41)
Sodium acetate•3H 0 (360 g) was dissolved in a Acetate buffer: 2 solution of 67 m of glacial acetic acid in 500 ml of double distilled I water and made up to 1 I i ter.
Cyanoacetate buffer: KCN (0.01 M, 0. 2 ml) was added to 10 ml of acetate buffer .
3% ninhydrin solution: Ninhydrin {3 g) was dissolved in 100 ml of ethyl cellosolve {2-ethoxy ethanol).
Ninhydrin Test Procedure
To 1.0 ml of testing solution was added 0.5 ml of distilled water,
0.25 ml of freshly prepared cyanoacetate buffer and 0.25 ml of 3% ninhydrin solution. It wo-:. mixed well and placed in a boiling water bath for 15 minutes . It was removed from the bath and cooled in a water bath. 24
1. 2
I. I
1.0
o.e
0.7 Q) (.) c 0 0.6 ..Q '- 0 0.5 fl) _Q 0.3 0. 2 0.1 0 0.1 0.2 0.3 0.4 Q5 0.6 O. 1 0.8 0.9 1.0 1.1 1.2 Canavanine Sulfate (mg) Fig. 11. A standard curve of PCAF test for canavanine sulfate. 25 Then isopropanol-H 0 {2ml, v/v=50/50) was added and mixed well. 2 The optical density was recorded at 570 m)I. PAPER AND THIN-LAYER CHROMATOGRAPHY USED ----- One dimensional paper chromatograms were developed by the ascending technique. Canavanine sulfate and the 50% alcohol extract were applied to Whiteman no. l paper using fresh solvent system of phenol : water (8:2). The dry developed chromatogram was sprayed with 0. 5% ninhydrin in n- butanol. The Rf value of canavanine was 0.45. The chromatogram of the 50% alcohol extract gave two Rf values of 0. 45 and O. 78 and indicated trace impurities. The dry developed chromatogram was sprayed with I ml of PCAF solution in 10 ml of phosphate buffer which was characteristic of canavanine. They both had the same Rf value of. 0.46. A paper chromatogram of canavanine extracted from beans using the with 0. 5% ninhydrin solution same solvent system and spraying gave an Rf value of 0.45 which was identical with an authentic sample of canavanine. A two dimension chromatogram of a mixture of canavanine and the resi- I due eluted with 1 N HC l from the AG-50W-8X column us ing fresh phenol: water (8:2) for the first dimension, and butyric acid:butanol:water (2:2: 1) for the second drmension and spraying with 0. 5% ninhydrin solution gave two pur- pie spots . This showed thrtt there were at least two different compounds in the mixture. Residue eluted with 1 N HC 1 gave a negative reaction for the PCAF test. 26 Thin-layer chromatography was used to identify the amino acids from the glutamic acid feed. Glutamic acid· HC (5...ul, 2mg/ml) and the radioactive l compound (3194.7 dpm), which was eluted with lN HCl and had a negotive PCAF test, were applied to the thin-layer chromatogram plate. The p.Ja-te was an Eastman 6065 cellulose with a fluorescent indicator. A fresh solvent (100 ml) of t-butanol:formic acid:water (7: 1 .5:1. 5) was placed in the devel- oping tank with an old chromatography plate and the system was allowed to come to equilibrium. The plate was placed hlathe developing tank. After about six hours, the plate was taken out of the tank and dried in the hood overnight. The strip containing the radioactive compound was cut out. It was run on the strip scanner to determine the position of the radioactivity. The remainder of the plate was sprayed with 0. 5% ninhydrin· in n-butanol ° and heated on a glass plate in an 110 oven until the colOr was developed. · ·The R v�lue of gh.1tamic. add'�HCl was 0.49. The dominent radioactive . f : . , peak on the chromatogram fell on the area which corresponded to· that for glutamic acid. This area was scraped off and dissolved in ] ml of water. . \ c acid· HCl {100 mg) and 9 ml of H 0 The solution was filtered and glutami 2 were added to the filtrate. It was evap:>rated to dryness and a white residue ca�e out. The yield was 91 mg. Recrystallized glutamic acid (1 mg) was dis solved in 2 drops of water and added to 10 ml of the tofoene scintillator for counting. The radioactivity recovered was 2802.8 dpm (87.J'Olo) . • 27 A thin-layer chromatogram (Eastman 6060 precoated silica gel plate with fluorescent indicator) of canavanine and arginine using an EtOH:NH 0H 4 (7:3) so lvent system gave an Rf value of 0.58 for canavanine and 0.22 for arginine . Thin-layer chromatography was prepared with 0.07 ml of the radioactive compound eluted from an IR-4B column using an EtOH:N H 0H(7:3) solvent 4 system. It was run on the strip scanner. Only one radioactive peak was shown, and it corresponded to the arginine spot. Canavanine and all the remaining solution of the radioactive compound eluted from the IR-4B column were applied to the sil ica gel plate and devel- · oped in the EtOH: H {7:3) so lvent �ystem. The portion of t e chromatOgram NH4 o � which contained the radioactive co�pound was· covered and the canavanine por- fion was sprayed with 0. 5% ninhydrin solution in n-butanol. One spotappe ared. The area corresponding to canavanine was scraped off, disso lved in 4 ml of H20, and filtered . A portion of the fil trate (o .1 ml) was added to'10 ml of the toluene scintillator forco unting . There was no radioactivity shown. The rema ining area was scraped off and dissolved into 5 ml of H 0 and filtered . A portion of the 2 filtrate was added to 10 ml of the to luene scintillator for counting. A radio- activity of 3000 dpm was shown . Arginine0HCl (100 mg) was dissolved in the filtrate and absolute alcohol (5 ml) was added. The precipitate was filtered and the dry weight was 92 mg . Recrystallized arginine (1 mg) was dissolved in 1 ml of H o and 10 ml of the toluene scintillator for counting . O. 2 28 The radioactivity recovered was 2769.2 dpm (92 .3%) . ISOLATION OF CANAVANINE Dried Jack bean (500 g} was ground to powder with a blender and a mil l. It was poured into a 4 I . Erlenmeyer flask and 2 of 50% alcohol was I. added, then stirred vigorously by a stirring �tor at room temperature over night. The extracted solutio n was filtered by suction. The extract was con- centrated to 200 ml under reduced pressure keeping the temperature below ° 50 C. Cold alcohol (99%, 1500 ml} was added, resulting in a light yellow gum (45 g} . The gum was dissolved in 50 ml of double distil led water and the pH was adjusted to 3.58 with 6N H2S04. The so lution was treated with 100 g of flavianic acid in 200 ml of warm double distilled water and placed in the refrigeratorfor two days. The yellow precipitate of dicanavanine tate (107 g} was fil tered and dec.omposed flavianate·appeared . The precipi . with hot saturated Ba(O H} (700 ml) . The fil trate (pH 10�3} was acidified with 2 . 6N H O to pH 4.25 to remove the excess of barium ion. The so lution was f 4 evaporated to 230 ml under reduced pressure and decolorized with 2 g of charcoal. + A 2x30 cm co lumn of AG-50W-8X (H Form) was prepared by washing the resin well with double distilled water. The colorless so lution (6 ml) was carefully added onto the top of the column and passed through the column. was eluted with the following solvents: It 29 ml of double distil led water 100 ml of lN HCl lSl 200 ml of 4N HCl The flow rate was set forab out 20 drops per minute and the effluent was col- lected in 10 ml fractions. Then two 1 ml aliquots of every other tube · were evaporated to dryness in the oil bath. They were assayed by the ninhy- drin test and the PCAF test. (Figure 12) · All PCAF test positive tubes (70 ml) were collected and evaporated to - 10 ml under pressure. A lx40 cm column of IR-4B (OH Form} was prepared by washing it with double distilled water. The acidic solution was added onto the top of the column and allowed to drain in. It was eluted with 300 ml of do uble distilled water and collected in 10 ml fractions. Two 1 ml aliquots of every other tube were assayed by the ninhydrin and the PCAF tests. (Figure 13) All the PCAF test positive tubes were collected and evaporated to 0 syrup under reduced pressure and 45 C. The free canavanine was precipitated by the addition of 5 ml of absolute alcohol. The precipitate was washed with 0 0 alcohol twice. This gave 0.3653 g. mp 180-184 {lit. 182-184 ) , C H O _N Anal. calculated: C 34.09 H 6.85 N 31.80 Found: C 33.35 5 12 3 . 4 1 =, 6. 51 (w .c. 2) (lit. 7. 9) - H 6. 67 � 30. 12 (a<)� + = _ IR(KBr) 3330 cm -1 -1 -1 (N H), 3450 3180 cm (N H ) , 1358 1410 cm (C=NH), 3410 cm & 2 & {COOH) . A mixture of canavanine (0 . 200 mo les) and arginine (0 .26 moles} was run on the short column for the automatic amino acid analyzer. It showed that they were separated and that canavanine· came out first. The isolated � I ' J ,, I \ I \ I \ I \ I \ 2.0l- I I Q) (.) ' § " +I \ 1.5 ' .c I "- 0 I \ U) \ .c I 90 0 30 70 10 50 90 130 10 so 130 170 10 50 90 30 70 110 150 30 70 110 150 190 ...J� 4 N HCI IE- H20 + 1 N HCI 'I' � E ff I uent ·Volume (m I) Fig. A ninhydrin test and a PCAF test for effluent from AG-50W-8X column 12 CA> -o-represents ninhydrin test and -e-represents PCAF test. 0 00 2.0 Q) 0 c: CJ ..c id 1.5 � 0 "' ..c <( 1.0 0.5 0 . .. 30 70 110 150 190 230 270 50 90 130 170 210 250 290 10. H20(ml) Fig. A ninhydrin test and a PCAF test effluent from IR-4B column 13. for � (,.) represents ninhydrin test and � prepresents PCAF test. - 32 canavanine (0. 41..Mmofes) gave only a canavanine pea k on the short column forthe automatic amino acid analyzer. The method of isotopic dilution analysis was used to isolate canavanine from the glutamic acid feed. Canavanin e• H2SO 4 (200 mg) was mixed with ground Jack bean. Canavanine was extracted as previously described . DEGRADATION OF CANAVAN INE (F igure 14) Canavanine • H SO (50 mg, 0. 2838 m mo le . 2 4 s) was dissolved in 10 ml of doub le distilled water in a 50 ml flask and the pH of the solut.ion was adiusted to 7. 5 with 1 N NaO H • Arginase (25mg, 24 uni ts) was added to the solution. It was incubated at room temperature unti I it no longer gave a PCAF test. The enzyme was destroyed by placing the flask in a boiling water bath for 10 minutes. After centrifuging , the solution was filtered into a 50 ml flask and 1.5 ml of 5% glacial acetic acid solution of xanthydrol(0�25 g of xanthydro l dissolved in 3. 5 ml of glacial acetic acid) wa� added . (42) The contents were mixed thoroughly by closing the flask with a clean cork and shaking vigorously. It was al lowed to stand for one hour . The . dixanthydryl urea was filtered by suction and the filtrate was stored in the refrigerator. The dixanthydryl urea was washed with hot absol ute alcohol (10 ml) to remove the excess xanthydrol. It was recrystallized from glacial acetic acid. ° ° The yield was 13.8 mg (18.03%) mp 172 deep. (lit. 174 deep.) :2) solv syste A paper chromatogram using the phenol: H20 (8 ent m; gave an 33 0 II H+ + OCC. '/OHc . . ,H. C..., . fl . 0 coJ · + Fig. 14. Degradation of canavanine. 34 Rf value of 0. 704, the same value as canaline. Sodium citrate (25.46mg , 0. 0866 m moles) and citric acid (254.6 mg, 1.33 m moles) as buffer (43) were disso lved in 10 ml of H 0 in a 50 ml two necked round-bottom flask. Nin 2 � hydrin (200 mg, 1.12 m moles) was added first, then the filtrate of canaline. The mixture was heated in a boiling water bath for one hour and a half. Nitrogen gas went into the flask through the saturated Ba(OH) tube. The 2 co was swept out with N and collected by saturated Ba(OH) • The 2 2 2 BaC0 was centrifuged, washed with water, ethanol, and ether and 3 then dried by air. The yield was 35 mg (0. 195 m moles, 97%) METHOD OF FEEDING PLANTS AND GROWTH OF PLANTS Seeds of the Jack bean were planted in the soil outdoors. After about three months, the plants flowered and set pods. After the pods became sufficiently large (2. 7 cm length seeds in pod), about one month, 14 DL-methionine-1- c (0 . 1 me/ml) was administered to the lants by means p . of cotton thread inserted through the stem near the first node. Five days after feeding the isotope, the fruit was harvested (fresh weight 923 g). Seeds of the Jack bean were planted in the soilof pots outdoors. The plants flowered after about three months. They were moved into the green house due to the weather. When the pods had grown to about 10 cm in 1 4 length, DL-glutamic acid-J- c (75 me/ml) was administered to the plants 14 the same as the methionine-1- c was . The fruit was harvested on the fourteenth day . The fresh weight was 17.6910 g. 35 LITERATURE CITED 1. M. Kitagawa and T. Tomiyama, Proc. Imp. Acad . {Japan) , �' 380-3 (1929}, cf. C. A. 24:373 M. Kitagawa and T. Tomiyama, J. Biochem. (Japan), 11, 265-71 i . (1929), cf. C. A. 24:1131 3. M. Kitagawa and H. Yamada, J. Biochem. (Japan) , �' 339-49. J. Arg. Chem . Soc ., (Japan} , �' 1135-40 (1932), cf. C. A. 27: 1020 4. M. Damodaran and K. G. A. Narayanan, Biochem. J. , 39, 1740 (1939) 5. E. A. Bel l, Biochem. J. , 75, 618-20 {1960) 6. B. A. Birdsong, R. Alston and B. L. Turner, Can. J. Bot. 38, 499-505 {1960) 7. S. E. Williams and G. E. Hunt, Planta {Berl.), 77, }92-202 (1967) 8 . E. A. Bell, Biochem. J. , 97, 104-1 11 (1965} 9. Dictionary of Organic Compounds, ..!_, p. 546, Oxford University press, New Yo rk, (1965) 10. M. Kitagawa and S. I . tv\onobe, J. Biochem. (Japan), �' 333-43 9 (1933) cf. C. A. 28: 1021 11. M. Kitagawa and S. I. Monobe, J. Arg. Chem. Soc. (Japan), �' 6 845-58 (1933) cf. C. A. 28:2678 ,' 36 12. M. Kitagawa and A. Takani, :J. Biochem. {Japan), 23, . 181-5 (1936), cf. C. A. 30:48 183 13. M. Kitagawa, J. Biochem. (Japan),.. 24, 107-12 (1936), cf. C. A. 4 30:8162 . 14. D. D. Nyberg and B. E . Christensen, J. Am. Chem. Soc., 79, 1222 (1957) 15. M. Kitagawa and A. Takani, · J. Arg . Chem. Soc. (Japan), .!.!_, 1077- 6 82 (1935) I cf. A. 30:3409 c. 16. J. Gulland and C. Mo rris, · M. J. Chem.. Soc. , 763-69 (1935) 17. M. Kitagawa and J. Tsukamoto, J. Biochem. (Japan), 26, 373 (1937), cf. C. A. 31:4275 2 18. M. Damodaran and K. G. A. Narayanan, Biochem. J., 33, 1740 -2 (1939) 19. K. I. Konobu, Ya kugaku Zasshi; 82, 1671-3 (1962), cf. C • . A. 59: 1749a 20. A. W. Naylor, Science,. 154, 425 (1966) 21. G. A. Rosentha l, Plant Physiol., 47, 209-2 11 {1971) 22. G. A. Ro sentha l, Plant Physiol., 46, 273-276 (1 970) 23. J. H. Johnstone, Biochem. J. , 64, 21p, {1956) 24. J. Walker, J. Biol. Chem. ; 204, 139-146 (1953) 25. B. Tschiersch, Diss. z. Martin-Luther University, Holle-Wittenberg, Math. Not. Reihe, 10(5), 1125-7 (1961), cf. C. A. 59:119009 37 26. R. P. Warren and G. E. Hunt, Plant Physiol. , s-7 (1969) 44, 27. Vo n R. Topfer, J. Miersch and H. Reinbothe . Biochem. Physiol. Pflanzen Bd., 161, S. 23 1-42 (1970) 28. Y. Knobler and M. Frankel, J. Chem. Soc., 1632 (1958) 29. E. Leete, J. Am. Chem. Soc ., 86, 3162 (1964) 30 . J. Walker, Comparative Biochemistry of Arginine and Derivatives, Ciba Foundation, Study Group No. 19, p. 58 31. A. White, P. Handler and E. L. Smith, Principles of Biochemistry, p. 479 (1964) 32. D. M. Mikhlin, C. R. Acad . Sci. , U. R. S. S., 20, 149 {1938) 3 cf. C. A. 33:2561 33 . W. H. Elliot and E. F. Gale, Nature 161, 129 (1949) 34. N. Grossowicz, E. Wainfan, E. Borek and H. Waelsch, J. Biol. Chem., 187, 111 (1950} 35. H. Waelsch, P. Owades, E. Borek, N. Grossowicz and M. Schou, Arch. Biochem., 27, 237 (1950) 36. W. H. Elliot, Biochem. J. , 49, 106 (1951) 37. G. C. Webster , Plant Physiol., 28, 724 (1953) 38 . G. C. Webster, Plant Physiol., 28, 728 (1953) 39. G. C. Webster, Arch. Biochem. Biophys. , 47, 241 (1953) 40. W. R. Fearon, Analyst, z.!_1 562 (1946) 41. H. Rosen, Arch. Biochem. Biophys. , 67, 10 (1957} 38 42. B. Harrow et al., Labora tory Manual of Biochemistry, fifth edition, W. B. Saunders Company, p. 134 (1960) S. P. Co lowick and N. Kaplan, Methods in Enzymology, 43. Oo vol . IV, Academic Press Inc., New Yo rk, N. Y. 1957, p. 71 1