Ill HM 146 227 THS A STUDY OF AN QXALACETATE DECARBOXYLASE iN CUCURBIT SEED GLOBULBNS AND THE RELAHON OF BIQTIN TO ”5 AC'FIVITY Thesis far the Emma of M. 5. MICHiGAN SYATE COLLEGE Sfeswar? Angiin Brown “M? \ -_-Q .,9'.“-: 1 ' J! ‘1‘: . 2 I“ r, , - (d ‘ ~‘IJ W 5% -‘-.‘v- J‘IJ‘VI. WV} o ' : f 31-; _ m w i; .. ‘ fly)", a ' w. ‘- (- eff. ‘ l r -.:.“'”.v.: I": ‘2'. ‘ i . 2f. 3...“: '2‘... J 3.1“ n -_ .-.. a This is to certify that the thesis entitled A STUDY OF AN OXALACETATE DECARBOXYLASE IN CUCURBIT SEED GLOBULINB AND THE RELATION OF BIO'I'IN TO ITS ACTIVITY presented by Stewart Anglin Brown has been accepted towards fulfillment of the requirements for ALE—0— degree in .2 Loohemietry Maw Major professor Date M v-r—‘r‘ ‘— ~— ——'——'——— --.v-..-— .—.v— —— —-——-— ‘V ._ —'—_.v .-— F ~ -- —.—.'~v. — — —. ...~ 1.. '— v-v‘»v ._ . I ~ ’2‘ .I' a A BTUDY OF AN WHITE DECARBOXYLASE IN CUCURBIT SEED GLOBULINS AND THE RELATION OF BIOTIN TO ITS ACTIVITY By BTW? ANGI-Il! BROWN A “H1313 Submitted to the School of Graduate Studies of Michigan state College of Agriculture and Applied Science in partial fulfillmb of the requirunents for the degree of msm OF SCIERCE Deper‘tmmt of Ghenietry 1949 lbl/vA/lu 8237‘? 216919 gnmsmmr The author wishes to express his thanks to Doctor R. U. Byerrun and Professor C. D. Ball for suggestions and enomragement git'en during the work on this problen. Gratitude is also due to the other faculty members of the biochemistry section for any assistance rendered. and to Mr. Harold Taylor of the Michigan Departmnt of Health laboratory in Lansing for his kindness in running the electrophoretic patterns. t.....” Otttfil *t*. it t C ON '12? TTS IITTlZCT-‘UCTIOI:COOOOOIOO0.0.0....000.......OOIOOOOOOOOOOOOOOCO00.... l v ,fim‘fi ”Q. IIIULL/IQIWLLeeeaeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeoeeee 2 EKPERIIEETAL..................................................... 4 haracterization of the Enzyme......................... 5 Effect of Salts on Enzyme Activity and on the Electrophoretic Pattern of the Globulin........... 6 Biotin Analysis of the Globulin Samples................ 7 Addition of Biotin to the Enzyme System................ 9 Removal of Biotin from the Enzyme System............... 12 DISC-1:533:01: 11;? COI:CLUSIO:L:SCOOOCOCCO0.0...OOOCOCOOOOOC.OOOOOIO...C l4 w-{fim‘tpvv T" 16 .L $J-JLLHL’IUHQOOOO.00...000......00.00.000.90000000000000.00.000.000. *ihk INTROWCTION For several years past considerable attention has been focussed on the elucidation of the biochemical function of biotin. Inasmch as several B-complex vitamins have been demonstrated to possess a eoensyne mnction, it has been felt that there nae a possibility of assigning a similar role to biotin. Work undertaken to discover the ooensyne function of biotin has provided a considerable amount of indirect evidence, but as yet it cannot be said that any conclusive proof has been obtained. i'he'present work was undertaken in an ef- fort to shed sons further light on the possible coensyns function of this vitanin. -1- geronxcn. Pilgrin.and coduorkers (1) in 1942 found that biotin, together with pantothenic acid, was essential for normal pyruvate metabolism, and suggested that these titanins were components of the particular enzyme systems involved. In.1947 tardy, Potter and Edvehjen.(2), working with Lactcbacillus arabinosus, found that in.a nediun.deficient in biotin and aspartic acid, the addition of exalacstate caused in- creased growth. They advanced the theory that biotin was necessary for the synthesis of oxalacetate by the condensation of pyruvate and 002, and that the oxalacetate could then be transaminated to aspartic acid in the absence of biotin. Stokes and coduorkers (3) also found, in their studies of lactic acid bacteria, that these organisms could syn- thesise aspartic acid only in the presence of biotin. These findings 'Iere confirmed by Potter and livehjen (4). Shire and Rogers (5) have suggested that biotin.is also involved in.the'biosynthesis of aéketo- glutaric acid from.cxalsuccinic acid, at least in.B. codi. Further work on this organism has best reported by Lichstein and cc-workers (6, 7). They found that if the organism was held at pH 4 in a phos- phate buffer at 31‘ for one-half to one hour there was a decrease in 002 production from aspartie, nalic, and onalacetic acids, as well as a decreased rate of deeminaticn of several amino acids. Adding biotin was reported to restore the activity. and in the case of the desalina- tion reactions adenylic acid also increased the reaction rate. Axelrod and co-workers (8), however, have been unable to demonstrate any reactivation of E. coli by biotin, and Lichstein (9) has recently indicated that the discrepancy is due to differences in the culture media used. Hence it does not seem advisable to regard this effect of biotin as definitely established. EXPERIMENTAL In attempting to study the effect of biotin on caalacetate decor- boa-ylases, it seemed advisable to select as pure an enzyme system as possible. In this respect the cuourbit seed globulin ensyne reported by Vennesland and Felsher (10) appeared pronising, as the globulin is crystalline and can be further mrified by recrystallisation. The globulin used in these investigations were prepared in all cases from cucurbit (principally squash and pinpkin) seeds by students in the Departth of Chemistry at Michigan State College, following the method of Viokery _e-t_ _a_l_. (11). They were not specially prepared for this work. All samples were tested for enzyme activity, using the larburg apparatus to neasure the amount of CO2 liberated in the docs:- bcacylation of a substrate of onalaoetic acid. A solution or fine sus- pension cf the asyne was introduced into the Warburg flask with a pipette, together with 0.5 ml. of an acetate buffer of the desired pH. 1 0.1 ml. portion of a solution of oxalacetic acid (10 mg. per m1. 320; prepared by the method of Schneider (12) ) was placed in the side arn. Any other tutorials required in the individual «perineum were placed in the flask and water we added to mice a total of 8.0 ml. After a ten minute equilibration period at 30°, sore-tine readings were nde on the mnometer and the reaction was started by tipping the flasks to nix enzyme and substrate. Readings were then taku at convenient in- tervals during the time required for the reactim. Bone globulin w...) 00g EVOLVED ")0 00 (3 6C) 2C) L l l (I K) l5 2C> ENZYME (MGJ ' 313333 I - Effect gz'inzyne Concentration gg'hetivity Readings were taken 150 seconds after mixing enzyme and sub- strate. Values are corrected for Spontaneous decomposition of oxalecetate. 0.5 ml. of 0.1M acetate buffer, pH 4.0, (final R = 0.016); 1 mg. of oxelacetate in 0.1 ml.; 1 m1. of enzyme; water to make 3 ml. IOO '- 80 60 (2(3CM2 40 20 4 a. o 1 I 1 1 I 1 1 _ 3.5 4.0 4.5 5.0 5.5 pH FIJUiE II - ?elaticn of Enanfi Activity to p~ .e-n‘.’ 4‘. The enzyme was diluted 1:320 with water. .2302 741. of CGg/x:.g.of enzyme / hour (based on 20~minute readings). Values are corrected for spontaneous decomposition of oxelacetete. 0.5 ml. of 0.1M acetate buf- fer, pH 4.0, (final K = 0.016); 1 mg. of oxalacetatg in 0.1 ml.; 1 ml. of enzyme; water to make 3 ml. samples were found to possess high decarbcxylase activity, others very little. Since considerable difference in solubility we noted, this may have been due in part to denaturaticn. The majority of the investigations were carried out on a single sanple of pumpkin seed globulins. except where it me desired to compare the activities of two or more samples. Characterisation of the Ezyme Before beginning studies on the relation of biotin to enzyme activity it as necessary to establish the optimum conditions for the determinations. Figure I shows the relation of activity to enzyme concentration. It can be seen that when readings were taken at the end of 150 seconds the relationship is practically linear up to about 10 mg. of globulin. Beyond this point the substrate pre- sumably begins to be the limiting factor. Ra's it feasible to take readings earlier, the linear relation would undoubtedly hold at higher enzyme concentrations as well. In Figure 11 is shown the activity-pH curve for the enzyme. The optimn pH under the conditions used lies botwou 4.0 and 4.6. The globulin was found to be alncst completely in the form of a suspension at pH 5.0 and above. At a pH of 4.5, near the optimum, it was still only partly dissolved, whereas at 4.0 and below solution was appar- ently complete. These results show that the activity is not directly proportional to solubility, a finding which was subsequently confirmed 0 LS? 2.55 3.3: ’ FIGURE Ills Electrophoretic Pattern.g§_Pumpkin Seed Globulin Protein concentration = 1%. Buffer - 0.08M Ra acetate, 0.4M acetic acid. pH = 4.02. r/z = 0.08. Specific conductance = 0.00290 mhos. Time = 103 minutes. Current = 0.0198 amps. Potential grud- ient = 8.62 volts / cm. Descending boundary is shown. Numbers below firure represent migration in inches from the starting boundary in the given time. 5 Peek v x 10 Area 4: (from left) 1. 3.5 6 2 2. 4.8 247 92 3. 6.2 16 6 FIGURE IIIb - Electrophoretic Pattern 93 Punmkin Seed Globulin by Venneslend end co-Icrkers (13) efter the completion of this work. The electrophoretic pettern shown in Figures III (s) end III (b) represents e solution of the globulin in ecetete buffer. This pettern use run st the seals pH end ionic strength es use used for measurement of the enzyme ectivity. From celculstion of the erees under the peeks it hes been found thet the principel component is present to the ex- tent cf 92%, horses the mll peeks to the left end right represent, respectively, 2% end 6% of the total. The slow-moving component be nigreted 1.87 inches, the mjor component 2.55 inches, end the fest- noving component 3.51 inches from the sterting boundery. The centre peek is symstricel, suggesting thet the component inch it represents is pure. Effect of Selts on Enzyme Activity end on the Electrophoretic Pettern of the Globulin hrly in the investigation en effort ms undo to get the only!” into solution et pH 5.0 by the use of e dilute solution of sodium chloride. While it use found possible to dissolve nest of the globu- lin, en elnost totel loss of enzyme ectivity elsc resulted. This effect is not specific to sodium chloride, es it is elso produced by emcniun sulphate. Figure IV show totel inhibition of the ensyne shen in e solution only 0.33% in respect to emoniun sulphete, end per- tiel inhibition then the selt concentration us 0.013%. It as noted thet et the higher concentration on epprecieble pert of the globulin Its insoluble et pH 4.0. (p...) 00, evoweo I60 |20 " D 80 '- h ‘ ‘ 40 '- ”’— v” ’ A "’ —" v". I— v' . C o.”’ a” - J l l l I l l l l l L 0 300 000 900 I200 TIME (SECS) Ia”-"‘.’T3.;'—" IV - fif‘f‘ect of .‘rgronium Svlphate on F'Tn";.'“.=3 :"-°T-iV1tV. ego Enzyme was diluted 1:320 with water. 0.05 ml. of 0.4... acet- ate buffer, pH 4.0, (final 2.: a 0.016); 1 mg. of omlacntate in (3.1 3111.; l Lilo of enzyzw; water to make 3 ml. 0 - Normal enzyme. 4 - Ln..;,nr;a suspended in 0.313,? (1.711,)833“ ‘37; ( 0 - Enzym snapended in GAL .. Jae-3,. I - Boiled enzyme. Further light my be shed on this phenomenon by en emanation of Figures 'V (e) end V (b) . which show e second electrophoretic pettern nde on the globulin in the presence of sodium chloride. In this cese the ionic strength as minteined et 0.08 by substituting sodium chloride for sodium ecetete. Here it cen be seen thet the mo- bilities of et leest the tee tester-moving frections here inoreesed in conperison to the mobilities shoun in Figure III. The slow-moving component Ins diseppeered atirely, coinciding with the loss of enzyme ectirity. It my here been rendered insoluble by the sodium chloride, end in this cese there is the possibility thet the ectivity is con- centrated in this smll frection. Another possibility is thet the nobility of the slow-tearing component hes been increesed we in propor- tion to the mobilities of the other tso components. If this is the cese its nobility ney be similer to thet of the njor component. Sup- port is lent to this possibility by the esynetry of the njor peek. To test the possibility thet the ectivity is concentreted in one minor component of the globulin, prelininery experimedu were con- ducted in which the most soluble frection use removed by dissolving in ueter, end the leest soluble by frectionetion with sodium chloride. In neither cese did eny nnrked loss of eotivity result. Further work is indiceted to clear up this question. giotin Luelysis of the Globulin Semjles Severel epproeohes were tried in en ettenpt to relete the ectiv- ity of the omelecetete deoerboxylese to the biotin content of the [) :3J55 ‘LCN4 FIGURE Va Electrafloretic I‘attern 9.1: Pumpkin £28.23 Globulin ?rotein concentration = 1.6%. Buffer - 0.08M.Na acetate, 0.101.: acetic acid, 0.062.: Naci. pH = 0.97. We = 0.08. Specific conductance = 0.00408 mhos. Thus = 120 minutes. Current = 0.0230 amps. Potential gradient = 7.52 volts / cm. Descending boundary is shown. Numbers below figure repre- sent migration in inches from starting boundary in the given time. Peak v x 105 Area ” (from left) 1e " a e- 2. 5.8 426 95 3. 7.5 21 5 FIGURE Vb - Electrophoretic Pattern QEPLUIIPKI'LD. Seed Globulin IOO - o 1 1 l I 1 1 l 3.5 4.0 4.5 5.0 5.5 pJ1 FIGUfi; II - ?elation of Eréyvo Activity to pH .— The enzyme was diluted 1:32 with water. 9002 =fil. of Cog/ng.of enzyme / hour (based on 20-minute readings). Values are corrected for spontaneous decomposition of oxalacetate. 0.5 ml. of 0.1M acetate buf- fer, pH 4.0, (final R = 0.016); 1 mg. of oxalacetate in 0.1 ml.; 1 ml. of enzyme; water to make 3 ml. samples were found to possess high decarboxylase activity, others very little. Since considerable difference in solubility was noted, this my have been due in pert to denaturation. The majority of the investigations were carried out on a single sample of pumpkin seed globulins, except where it no desired to compare the activities of two or more samples. Characterisation of the Mayne Before beginning studies on the relation of biotin to ensyne activity it was necessary to establish the optimal conditions for the determinations. Figure I shows the relation of activity to ensy'm concentration. It can be see: that when readings were taken at the end of 150 seconds the relationship is practically linear up to about 10 mg. of globulin. Beyond this point the substrate pre- sumably begins to be the linting factor. Ice it feasible to take readings earlier, the linear relation would undoubtedly hold at higher enzyme concentrations as well. In Figure II is shown the ectivity-pH curve for the mm. The optima pH under the conditions used lies between 4.0 and 4.5. The globulin we found to be alnost completely in the forn of a suspension at pH 5.0 and above. At a pH of 4.5, near the optim, it was still only partly dissolved, whereas at 4.0 and below solution was appar- ently complete. These results show that the activity is not directly proportional to solubility, a finding which was subsequently confirmed 0 LB? 2.55 3.3: 5 FIGURE Illa Electrophoretic Pattern.g§.Punpkin Seed Globulin Protein concentration = 1%. Buffer - 0.08% Ea acetate, 0.4M acetic acid. pH = 4.02. N2 = 0.08. Specific ccnductancc a 0.00290 mhos. Time = 103 minutes. Current = 0.01?8 amps. Potential grad- ient = 8.62 volts / cm. Descending boundary is shown. Numbers below firure represent migration in inches from the starting boundary in the given time. Peak v x 105 Area ii (from left) 1. 3.5 6 2 2. 4.8 247 92 3. 6.2 16 6 FIGURE IIIb - Electrcmhoretic Pattern 93 Pumikin Seed Globulin by Vennesland and co-wcrkers (13) after the completion of this work. The electrophoretic pattern shown in Figures 1111 (a) and III (b) represents a solution of the globulin in acetate buffer. This pattern was run at the same pH and ionic strength as was used for measurement of the enzyme activity. From calculation of the areas under the peaks it has been found that the principal component is present to the ex- tent of 92%, vherees the sail peaks to the left and right represent, respectively, 2% and 6% of the total. The slow-moving component Ms migrated 1.87 inches, the mjor component 2.55 inches, and the fest- noving component 5.51 inches from the starting boundary. The centre peak is symetrioal, suggesting that the component iiich it represents is pure. Bfect of salts on Shayne Activity and on the Electrophoretic Pattern of the Globulin hrly in the investigation an effort was ends to get the enzyme into solution at pH 5.0 by the use of a dilute solution of sodium chloride. While it was found possible to dissolve most of the globu- lin, an elnost total loss of enzyme activity also resulted. This effect is not specific to sodium chloride, as it is also produced by emcniun sulphate. Figure IV show total inhibition of the ensyne when in a solution only 0.53% in respect to moniun sulphate, and par- tial inhibition when the salt concentration to 0.013%. It was noted that at the higher concentration an appreciable part of the globulin was insoluble at pH 4.0. -6- mt.) co, evowso I50 I20 '" - - ’ 4o .— fiv” "’ ’G” A ”’ v” o ’- 0 O"’ . ’0 o" - l I l 1 l l l l l l l 0 300 500 900 I200 TIME ( SECS.) T . v a A _ (- ‘“ T_" ~ .el 3“; IV - 7.?“001'. of .-.:*;'.onitm cu...-hate on mans: .-«.ctiVitv -_ ’— T. 4—4. Enzyme was diluted 1:320 with water. 0.05 ml. of 0.131? acet- ate buffer, pH 4.0, (final X a 0.016); 1 mg. of ozalacntata in 0.1 ml.; 1 2.1. of enzyme; water to make 3 ml. 0 - Normal enzyme. 4 - Enaysae suspended in 0.33,? Bridges“ pan-4 0 - Enzyme suspended in 0...“..0 0:3,)230“ I - Boiled enzyme. Further light my be shed on this phenomenon by on mainstion of Figures 'V (s) nnd V (b) . which show e. second oleotrophoretio pattern nde on the globulin in the presence of sodium chloride. In this cese the ionic strength as maintained st 0.08 by substituting sodium chloride for sodium oootsto. Here it can be soon othst the no- bilitios of st least the two foster-moving fractions have increased in compsrison to the mobilities shown in Figure III. The slow-moving component his diseppeered entirely, coinciding with the loss of enzyme activity. It my have been rendered insoluble by tho sodiun chloride, end in this csso there is tho possibility thet tho scti'rity is con- contrstod in this smll trsction. Another possibility is that the nobility of tho slow-moving component has been inoroesod we in propor- tion to the mobilities of the other two components. If this is the case its nobility my be similar to thst of the njor component. Sup- port is lent to this possibility by the esymetry of the njor peek. To test the possibility that the ectivity is concentrated in one ninor component of the globulin, prolininery experiments were con- ducted in hid: the nost soluble freotion was removed by dissolving in inter, end the least soluble by froctimtion uith sodium chloride. In neither one did eny lurked loss of sctivity result. Further work is indiceted to clear up this question. giotin Analysis or the Globulin Bangles Borersl epprosohos were tried in on sttonpt to relate the ectiv- ity of tho oxelsootete dooarboxylsso to the biotin content of the -7- - 7"- I-‘Iu‘r it"s Va Electrcmoretic Pattern 91 Pumpkin 53333 Globulin Protein concentration 2 1.6%. Buffer - 0.02M Na acetate, 0.10;: acetic acid, 0.062.: NaCl. pH = 3.97. fig = 0.08. Specific conductance = 0.00408 mhos. Time = 120 minutes. Current = 0.0230 amps. Potential gradient = 7.52 volts / cm. Descending boundary is shown. Numbers below figure repre- sent migration in inches from starting boundary in the given time. Peak v x 105 Area .E (from left) lo "' - - 2. 5.8 426 95 ['0 H U‘ 3. 7.5 FIGURE Vb - Electrophoretic Pattern pimepkin Seed Globulin system. The first of those involved comparing the activities of several samples of enzyme and trying to correlate activity to biotin content. .i'ho biotin content of each sample was determined by micro- biological assay methods. it first the organism used was gotobacillus 23:1. The culture medium and technique were essentially that of Henderson and Snell (14), an automatic titration assembly being used for measurement of the growth by ascertaining the amount of lactic acid produced from D-glucose. After some weeks, however, it became very difficult to obtain consistently good results with this method, and it was abandoned in favour of that of snoll (15) using the growth rate of the yeast Baccharayces cerevisiao. The only essential modi- fications in the latter method were the substitution of an equivalent quantity of calcium pantothonate for B-alanino in the culture medium, and the use of a turbidouetric method for growth measuremuts. Road- ings were nude in a Helligo-Diller colorinotor using a 660 my filter. This yeast growth method proved entirely satisfactory. In preparing the tutorial for assay, 0.1-1.0 g. of the globulin were hydrolysed with a large excess of 3N hydrochloric acid under fif- teen pounds pressure for one hour. The resulting solutions were evapo- rated to dryness on the steam bath to drive off most of the acid, and any retaining was nutralisod with 0.211 sodium hydroxide to the pH of the culture medium used. Since biotin is apparently resistant to strong mineral acids (16) little biotin is probably lost by this techni- que. In any case. the method would appear to be valid for direct com- parison of samples. Table I Rolatiai Between My: Activity 2d Biotin Content sums 310m ACTIVITY (diluted 1:15) (,ug./ 3..) (pl. 002/ 600 secs.) 1. 0.044 21 2. 0.017 95 Activity was determined at pH 5.0 in 0.1 M acetate buffer using standard Warburg technique as previously described. Attempts to correlate biotin content and enzyme activity proved unsuccessful. i'able I shows a typical comparison of two globulin preparations as to biotin content and enzyme activity, using the 3232:} method; in this case the sample with the higher activity had the lower biotin content. In another assay, using the yeast method, two samples having about the same activity showed a great difference in biotin contmt. These results do not necessarily eliminate the possibility of a ccensylno function for biotin, though, since part of the biotin my be combined with or adsorbed by a non-enzyme fraction of the globulin, which already has been shown to be impure. Addition of Biotin to the Enzyme Busters If biotin were indeed the coensyme for the seed globulin enzyme, two possibilities remained to be considered. (1) its little biotin may be present for minim activity of the enzyme; in this case add- ing biotin should result in greater activity. (ii) Sufficient biotin, or even an success, may be present; here, since biotin is assulsed to be the limiting factor, removal of more than a critical amount of biotin would result in lowered activity. Table II Effect 35 Added Biotin 2 2523 Activity ,uL. 002 EVOLVED (1200 sec.) 1. Enzyme (diluted 1:320) 49 2. Enzyme (diluted 1:320) +20 pg. of biotin 47 Activity was determined at pH 6.0 in 0.1 M acetate buffer using standard Warburg technique as previously described. Table II shows the effect of adding biotin" to pumpkin seed glo- bulin on its enzyme activity. There is obviously no significant dif- ference. The results reported by Vennesland and coworkers (13) are. in accord with this finding. It was thought worth while, nevertheless, to investigate the oxa- lacetate decarboxylase activity of sprouting seeds, since this activ- ity night be expected to reach a minus when the seed was actively metabolizing carbohydrate. In addition, it 1... thought possible that if biotin combines with the globulin protein enzymtically the neces- sary snsyne might be present in the sprouted seed to catalyze the attachment if biotin were added to the system. Squash seeds wens, ' The sample of biotin used was obtained from General Biochemicals, 1110., in crystalline fern. therefore, sprouted at room temperature in contact with damp paper towelling until sprouts at least 1 cm. long had developed. The seed coats were then removed and the reminder of the seed was ground with water in a Potter-Blvehjem homogeniser (17). The resulting milky fluid as used for determining enzyme activity. The foregoing opera- tions were carried out rapidly in chilled containers. Unfortunately, in such an impure preparation there were complicating factors, the chief of which was a large oxygen consumption. Because of these, it was not found possible to obtain consistent results. A typical exam- ple is shown in Table III, where it can be sea that there is a wide discrepancy between the results obtained from one preparation to the next. Running the reactions under 95% H2 - 6% 002 produced better checks, but even under these conditions, when the seeds were ground in a biotin solution instead of in inter, no reproducible effect could be found. The work with sprouted seeds was therefore abandoned. TABLE III 9325! in the Munster Read¥ Caused 111% the Action of Sprouted 833a“ wear—T— gemegn . um mfg- "" DETERMINATION PREPARATION UMBER w _ 1. z. 3. U41- ) (H1. ) ()41- ) 1e - 83 0 II 43 z. ‘- 90 t 17 n ‘0 Deter-intions were ads in 0.1M acetate buffer. The change in volume is at the end of 1800 seconds, corrected for flash constant. Honogenate was prepared by homogenizing 1 weight of seeds with 5 weights of water. Determination number indicates duplicate sampl. of the same prepara- tion -11- Resoval of Biotin from the mgyme System To remove biotin from the enzyme system use was made of the fact that the protein avidin, found in egg ilite, combines quantitatively with biotin in vitro (18). Figure VI shown some results obtained using egg white. In the presence of norml egg white an inhibition of about 40% was consistently obtained. Hwever, on dialysing the egg white for twenty-four hours it we partially precipitated, and the inhibition was reduced to about 10%. The behavior after dialysis would suggest that the inhibition was due, in part at least, to salt present in the undialyzed material, and not entirely to avidin activ- ity. It was found, though, that boiled and homogenized egg white inhibited only slightly, a fact which is difficult to reconcile with the previous results, since in the latter case the salts should still be present in the hanogenate. In view of the uncertainty involved in the use of crude egg white it was felt that a concentrated preparation of avidin would be neces- sary for any conclusive results. Accordingly, attempts were mde to obtain such a preparation from egg white. Hethods recon-sanded by hkin, Snell, and William (19) and by Woolley and Longsworth (20) were tried, but in each case no preparation could be obtained with any avidin activity assayable by the yeast method (19). The assay method was checked against egg white and found to be valid. In three attempts to fractionate the proteins soluble in 2% amonium sulphate, by the method of win _e_vt_ _a_l_., no separation could be achieved. Using -12- Mn.) 0 O T co, evoweo I40 I20 "' I00 '- O O I n c> I 20 I— Z / ’"fi—"’ 0 200 400 600 800 I000 TIME (5608.) ”Cw-e. Fiend}: VI - Effect of E"? White on Enzyme fiat-tiling; Enzyme was diluted 1:320 with water. 0.5 ml. of 0.11:? acet- ete buffer, pH 4.0, (final Li = 0.016); 1 mg. of oxelecetete in 0.1 ml.‘, 1 1711. of enzyme; water to make 3 ml. 0 - Normal enzyme. 4 - Enzyme 4* 1 ml. of normal egg white. 0 - Enzyme + l ml.of 8.53., white dialyzed against tap- and distilled water for 24 hours. 0 - Boiled enzyme. the Woolley and Longsworth method, the proteins could be fractionated, but still no avidin activity resulted. Snell (21) has stated that to be certain that the avidin has behaved as it should, it is necessary to perform an assay after every step of the concentration. Through the courtesy of Hoffman-LaRoche, Inc., an active sample of avidin assaying 2300 units per g. was obtained. (A unit of avidin will inactivate 1 pg. of biotin.) In ascertaining its effect upon the enzyme activity, 5 mg. of the avidin us finely suspended in 100 m1. of water. Part of the avidin apparently dissolved, and the rest re- snined suspended in the eater. This mixture Ias diluted 1:10, and a 0.5 ml. portion of the diluted sample gs added to the buffered enzyme solution in a Warburg flask. 0n the basis of the biotin assay of the globulin sample, there was at least 370 times as such avidin as would be required to inactivate all the biotin present. The avidin and biotin were allowed to stand in contact for abmit an hour, and the determination was then carried out as before. In Figure VII it can be seen that the addition of avidin produced no significant change in the activity of the myme, within the limits of error of the deter- mination. The inhibition observed in the case of undialyzed egg white mist, therefore, have been caused by salts present in the egg white. I40 '- I20 '- m 5 o o l l (:0, evowso ( pL.) CI 0 40 20 l L L I 0 200 400 600 800 1000 I200 TIME (SECS.) F1013 VII - Tim‘eat ____‘,_‘_ Pvt-din 3.3 114313 Aotivity Enzyme was diluted 1:320 with water. 0.5 ml. of 0.13.: acet- ate buffer, pH 4.0, (final Mh= 0.016); 1 mg. of oxalacetate in 0.1 1711.; 1 m1. of enzyme; water to make 3 .ml. A - Nonrxal enzzme. O - Enzyme + 2.5}15. of avidin. D - Boiled enzyme. DISCUSSIGI AND CONCLUSIONS Several attempts have been made to relate the onlacetate decar- bcnylase activity of cucurbit seed globulins to their biotin content, without success. The activity bears no consistent relation to the amount of biotin naturally present in the globulins. Adding biotin to the system results in no increase of enzyme activity, and under the conditions tried no inactivation of the enzyme could be obtained by adding avidin. It appears that two possible conclusions may be drawn from these results. (i) It is still possible that biotin, presart in success, may possess a ccenzyme function for this enzyme. If this is true, we are forced to conclude that the union of biotin with the enzyme is of such a nature as to nice impossible its union with avidin. This is not an unreasonable hypothesis, since both the enzyme and avi- din are proteins, and might logically be expected to combine by way of the same functional group on the biotin molecule. Hence if some group such as the carbonyl of the biotin were linked to the enzyme protein, this group would not be available for combination with any avidin that us present, and the latter would produce no inactivation. (ii) The alternative conclusion is that biotin does not function as a ccenzyme for the cucurbit seed globulin deoarboxylase. If this is so, it would argue for the possibility of a different mode of action as contrasted with similar enzymes in microdrganisms, since such of the ~14- evidence in the latter case favours such a ccenzyme function for biotin. The inactivation of the mm by salts is a phenomenon that de- serves further study. It is possible, on the basis of the electro- phoretic patterns, that the inactivation may be accomplished by precipi- tating out a small active fraction of the globulin. 0n the other hand, it is novel that the activity of an enzyme is closely connected to the charge it carries, and if the mobility of a fraction is changed it is an indication of an altered charge. Thus it is quite conceivable that the salt nay exert its effect by altering the charge on the active compon- ent. Further investigation is needed to determine the exact mechanism of this salt inactivation. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) REFERENCE Pilgrim, F. J ., Axelrod, A. E. and Elvehjem, C. A... ”The metabolism of pyruvate by liver from pantcthenic acid-wand biotin-~dsficient rats", J. Biol. Chem., 332, 237-40 (1942). Lardy, H. A., Potter R. L. and Elvehjem, C. A., “The role of biotin in bicarbonate utilization by bacteria“, J. Biol. Chem., 169, 451-2 (194?): etch“. J. Its, “”611, A. “d Gum”.' 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