Biotin Vitamin

ISOLATION BY OF BIOTIN (VITAMIN H) FROM HOFMANN,t University LIVER* AND VINCENT DU VIGNEAUD, KLAUS DONALD B. MELVILLE1 of Biochemistry, New York AND (From the Depurtment Cornell City) GYORGY Medical College, Downloaded from www.jbc.org by on August 30, 2009 PAUL (From the Babies School of and Childrens Hospital, and the Department Medicine, Western Reserve University, of Pediatrics, Cleveland) (Received for publication, June 6, 1941) In the course of our work on the isolation of vitamin H, the antiegg white injury factor, it began to be suspected that vitamin H might be identical with biotin, a yeast growth factor, which had been isolated as the methyl ester from egg yolk by Kijgl and Tennis (2), and with coenzyme R, a growth and respiration factor for rhizobia (3). All the evidence we were able to adduce, experimentally and from the literature, on distribution, stability, solubility, and various other chemical and physical properties favored the identity of vitamin H with these two factors. The similarity between biotin and coensyme R has been pointed out by other workers (4, 5). Our comparison of the biotin, coenzyme R, and vitamin H activities of electrodialysate fractions of our vitamin H preparation showed a striking parallelism in the distribution of the three activities. On the basis of this work we suggested (6) * A preliminary report of this work has appeared (1). In this cooperative research reported here, the Cornell group has been responsible for the chemical work and for the biotin assays, and the Western Reserve group for the vitamin H assays. The authors would like to express their appreciation to W. 0. Frohring, president of the S. M. -4. Corporation, for his interest and genuine cooperation. t Dr. Hofmann’s participation in this work has been made possible by a fellowship granted to him by the Societ,y for Chemical Industry, Basel, Switzerland. 1 S. M. A. Corporation Fellow. 643 644 Isolation of Biotin that vitamin H, biotin, and coenzyme R “are either identical or indeed closely related compounds.” Shortly after the above work was presented, we were able to test the possible identity of biotin with vitamin H by vitamin H assay of a solution containing 150 y of crystalline biotin methyl ester kindly placed at our disposal by Professor Kogl. On the basis of the ratio between the biotin activity of Kogl’s material and the biotin activity and vitamin H activity of other preparations we had studied we were able to predict that the biotin preparation from Kiigl should possess a vitamin H activity of 10,000 units per mg. (The vitamin H unit is the minimum daily amount of substance, injected for 30 days, necessary to bring about complete disappearance of egg white injury symptoms in rats.) As reported (7)) this prediction was borne out by vitamin H assays of the Kogl sample on rats. Since our vitamin H concentrates were prepared from liver and since Kijgl had isolated his biotin from egg yolk, it was necessary for us to attempt the isolation from the liver concentrates of the substance possessing vitamin H activity and to determine whether or not the properties of the pure substance were the same as those reported for biotin by Kijgl and Tennis. The starting material for the present investigation was a vitamin H liver concentrate supplied by one of us (P. G.) which had been prepared from the alcohol-insoluble fraction of beef liver by high pressure hydrolysis, precipitation of the inert material with alcohol and acetone, and precipitation of the active substance with phosphotungstic acid, followed by decomposition of the precipitate with barium hydroxide (8). The vitamin H activity of the resulting solution was 25 to 35 units per mg. of solids and 1000 units per cc. Each step in the fractionation of this liver concentrate given in the present paper was followed by biotin assays by a modification of the yeast growth method of Snell, Eakin, and Williams (9). Comparative assays for biotin and vitamin H activities of many of the intermediate fractions as well as the final product demonstrated again the parallelism of the two activities. One of the final steps in the lengthy fractionation procedure used by Kiigl and Tijnnis was the conversion of biotin to a biologically active “ester-base” by esterification with acidic methanol. Esteri- Downloaded from www.jbc.org by on August 30, 2009 du Vigneaud, Hofmann, Melville, Gyiirgy 645 fication of our crude preparation was carried out in order to obtain the active material in a form which would be soluble in organic solvents and which might lend itself to purification by chromatographic adsorption procedures. By adsorption of the crude ester from chloroform solution on aluminum oxide and by elution with acetone, the potency of the material could be raised from 25 to 50 vitamin H units per mg. to 1000 to 2000 vitamin H units per mg. A second adsorption on aluminum oxide and elution yielded material of a potency of 3000 to 6000 vitamin H units per mg. The eluate was evaporated and the residue taken up in chloroform. The solution was extracted with dilute HCl, and the extract was then evaporated to dryness. Downloaded from www.jbc.org by on August 30, 2009 FIG. 1. Biotin methyl ester. X 150 This preparation was esterified and the ester hydrochloride which was formed was converted into the free ester. Crystals were obtained which melted at 154-158”. By repeated crystallizations from a mixture of methanol and ether a product of constant melting point and biological activity was obtained which melted sharply in the Kofler micro melting point apparatus at 166-167” (uncorrected). Sublimation in ~~CUO followed by crystallization from a mixture of methanol and ether did not change the melting point, crystalline form, or biological activity. The substance crystallized, as shown in Fig. 1, in long, thin, plate-like needles from the methanol-ether mixture. From a chloroform-petroleum ether mixture it crystallized in needles as described by K6gl and TGnnis. The biological activity and melting point of the pure biotin methyl ester were confirmed by several separate isolations. 646 Isolation of Biotin Using the yeast growth method, we have compared the biotin activity of our purified crystalline material with the biotin activity of the vitamin H preparation (34 vitamin H units per mg.) that we had used as an arbitrary standard in our previous communications (6, 7). Expressed in terms of vitamin H units, the various preparations of purified product that we have made have all consistently yielded, by the yeast growth method, the high value of 27,000 (&lo per cent) vitamin H units per mg. Direct vitamin H assays of our crystals, made on rats by the curative method, were in agreement with this high potency. From 5 liters of the liver concentrate (8) we have been able to isolate 70 mg. of pure biotin methyl ester, which corresponds to a yield of 38 per cent based on our starting material. The analytical values we obtained for the pure crystalline compound, as shown in the experimental part, agree most closely with the empirical formula of CnH,,03N$. Molecular weight determinations by the Rast method with various solvents were not entirely satisfactory. However, all the results obtained with borneol indicated t,hat the empirical formula as derived from the analytical data on the basis of 1 sulfur atom in the molecule is unquestionably correct. Analyses of several crystalline derivatives and degradation products, which will be described in a later publication, likewise support this formula. This formula is in agreement with that given by K6gl (lo), although it should be pointed out that K6gl gave no analytical data. The melting point for the preparation from which K6gl derived this formula was not given; so one must therefore assumethat his previously published melting point of 148” still applies. It is to be noted that the melt’ing point of the pure biotin methyl ester obtained by us is 18-19” higher than the value reported by Kiigl and Tijnnis, and no explanation for this is as yet apparent. The similarity, howcvcr, in solubility, in crystalline form, and in composition leads us to believe that the compound isolated by us from liver is identical with that isolated by Kcgl and Tiinnis from egg yolk. As WChave stated, the solution of crystalline biotin methyl ester supplied us by K6gl showed a potency of 10,000 vitamin H units per mg., as compared with the value of 27,000 vitamin H units per mg. found for our preparations both by comparative assays Downloaded from www.jbc.org by on August 30, 2009 du Vigneaud, Hofmann, Melville, Gyiirgy 647 for yeast growth and for anti-egg white injury activities.1 However, we do not know the melting point or purity of this particular preparation of Kogl. We therefore do not wish to stress this difference in degree of potency. It is within possibility that the Kijgl sample may have suffered loss of potency from the time it was made up until it was assayed here. We can therefore simply report the activity of it as we found it. Direct comparison of the crystalline compounds is, of course, desirable and we hope that future circumstances will permit such comparison. EXPERIMENTAL Downloaded from www.jbc.org by on August 30, 2009 Method of Assay-Biotin determinations were carried out by a yeast growth method which is essentially that of Snell, Eakin, and Williams (9), except that Fleischmann Strain 139 of Saccharomyces cerevisiae was used as the test organism and turbidity measurements of yeast growth were made in the Klett-Summerson photoelectric calorimeter, with a blue filter (Klett No. 42). This instrument is equipped with a logarithmic scale, so that the turbidity readings observed are a linear function of the yeast growth. Each sample to be assayed was made up in a series of decimal dilutions of 1 and 0.3 cc., involving some twelve dilutions over a millionfold range. By plotting the resulting turbidity readings against the logarithm of the concentrations used, an S-shaped curve was obtained for each sample that was approximately linear over a considerable portion of the middle range. Comparison of the interpolated concentration at which half maximum growth occurred in the assay sample with the corresponding concentration obtained in the same manner with a solution of known vitamin H activity permitted calculation of the vitamin H equivalence of the sample being assayed. Isolation Procedure, EsteriJication-500 cc. of the vitamin H concentrate from liver mentioned in the introduction were concentrated to dryness in vacua and kept at 100’ for 1 hour. The 1 As mentioned in the preliminary report of this work (1) the above ratio was likewise found for the activities of the two preparations in promoting the growth of Rhizobium trifolii (coenzyme R activity) as determined by Professor Dean Burk and t,he growth of Clostridium butylicum as determined by Dr. D. W. Woolley. Isolation of Biotin gummy residue was then taken up in 200 cc. of absolute methanol and was again concentrated to dryness. 200 cc. of absolute methanol containing 5 per cent of dry HCl gas were then added and the solution was refluxed for 1 hour. The methanol was removed in vacua and the residue was reesterified in the same manner. The solution was then concentrated to a thick syrup in vacua; the residue was cooled to 0” and was washed into a separatory funnel with 50 cc. of ice water. 200 cc. of ice-cold ethyl acetate were then added, followed by 30 gm. of solid potassium bicarbonate, and the mixture was shaken until all of the bicarbonate was dissolved. The water layer, which was alkaline to litmus, was then extracted three more times with 100 cc. portions of ethyl acetate. The combined ethyl acetate extracts were washed repeatedly with water, dried over sodium sulfate, and evaporated to dryness in vacua. The ethyl acetate fraction contained 80 to 90 per cent of the activity of the starting material and it had a potency of 50 to 100 vitamin H units per mg. The water residues and washings were combined, evaporated to dryness in vacua, and the residue was reesterified by the procedure outlined above. First Adsorption on Aluminum Oxide-Several ester fractions were combined and purified further by chromatographic adsorption. One typical experiment was carried out as follows: 18 gm. of the crude ester fraction, containing 1,500,OOO vitamin H units, were dissolved in 150 cc. of chloroform and the solution was filtered through a column of 50 gm. of aluminum oxide (standardized according to Brockmann). The column was washed with the various solvents indicated in Table I, each washing being collected separately and evaporated to dryness. The samples were then assayed. The results of such an experiment are summarized in Table I. All the fractions containing an activity of 1000 vitamin H units per mg. or better were combined and used for the next step in the purification. Second Adsorption on Aluminum Oxide-2.55 gm. of this concentrate derived from several preparations which had been put through the first adsorption step and which contained 2,585,OOO vitamin H units were dissolved in 50 cc. of acetone, and the solution was filtered through a column of 100 gm. of the aluminum oxide. The chromatograph was again fractionated by elution with mixtures of solvents as indicated in Table II. Fractions 343-V Downloaded from www.jbc.org by on August 30, 2009 du Vigneaud, Distribution Column elated with Hofmann, TABLE I Melville, in First No. Weight Gyijrgy 649 of Biotin Activity Fraction Chromatograph Activity Vitamin H units Zli3’ cc. w7. vitaminH units Chloroform Acetone “ 90% an01 10% “ iI ‘I Total + meth‘I “ ‘I 300 300 50 50 50 50 339-x1 339-VIII 339-1x 339-x 339-x1 339-x11 15,000 805 21 76 256 102 16,260 147,000 1,000,OOO 45,000 145,000 310,000 45,000 1,692,OOO 9.8 1242 2140 1910 1210 440 Downloaded from www.jbc.org by on August 30, 2009 ._..........................._..., TABLE II in Second No. Distribution Column &ted with of Biotin Activity I kaction Chromatograph - Weight Activity cc. Acetone “ anol 90% 10% “ ‘I I‘ “ “ “ “ ‘I ‘I CL I‘ 6‘ + meth“ ‘I ‘I I< L‘ ‘I “ “ LL L‘ “ “ 200 130 10 10 10 10 10 10 10 10 10 10 10 10 343-I 343-11 343-111 343-IV 343-v 343-VI 343-VII 343-VIII 343-1X 343-x 343-X1 343-X11 343-X111 343-XIV ml. Zamin H unit l,ooo B 622 583 82 72 52 45 42 37 30 89 65 68 76 50 1913 Inactive 700 80,000 178,000 162,000 220,000 220,000 200,000 312,000 240,000 162,000 120,000 60,000 -. 1.6 0 8.5 1110 3420 3600 5240 5950 6670 3500 3700 2380 1580 1200 Total 1,955,700 to 343-X111 were combined and used for the next step. Fractions lower in activity were likewise combined and purified further by chromatographic adsorption. 650 Isolation of Biotin Extraction and ReesteriJication-The combined Fractions 343-V to 343-X111 (504 mg. containing 1,814,OOO vitamin H units) were dissolved in 10 cc. of chloroform and the resulting solution was extracted fifteen times with 10 cc. portions of 3 N HCl. The combined HCl extracts were evaporated to dryness in vacua and the dry residue (474 mg. containing 1,812,600 vitamin H units) was refluxed for 1 hour with 50 cc. of methanol containing 5 per cent of HCl gas. The methanol was then removed in vacua and the residue dissolved in a few cc. of ice water. Saturated potassium bicarbonate was then added to the solution until it was alkaline to litmus and the solution was extracted with three 50 cc. portions of ice-cold ethyl acetate. The combined ethyl acetate extracts were washed with water, dried over sodium sulfate, and concentrated in vacua. During the concentration crystals of biotin methyl ester began to separate. The crystals were collected and were washed with ethyl acetate. The yield of crude material which melted at 155-160” was 35 mg. Purification by two crystallizations from a mixture of methanol and ether yielded a material that melted at 166-167”. The material was sublimed at 140150’=’ in a high vacuum (10v6 mm.) and recrystallized from methanol and ether. 25 mg. of colorless elongated plates were obtained which melted sharply at 166-167’. The compound is very soluble in ethyl alcohol, methyl alcohol, chloroform, and acetone, and is sparingly soluble in ether and ethyl acetate. It possesses a specific rotation of [a]E2 = +57” for a 1 per cent solution in chloroform. The compound has the following composition. CJMhN~S. Calculated. Found. C 51.14, “ 51.40, H 7.02, “ 7.19, N 10.84, “ 10.84, S 12.42, “ 12.16, OCH3 “ 12.0 11.5 Downloaded from www.jbc.org by on August 30, 2009 The authors wish to express their appreciation to Miss Eleanor Hague for carrying out the numerous biotin assays, to Dr. J. R. Rachele for the microanalyses, and to Miss Catharine S. Rose for her assistance in the vitamin H assays. SUMMARY A procedure for the isolation of the methyl ester of biotin min H) from liver has been described. The compound (vitaafter du Vigneaud, Hofmann, Melville, Gyijrgy 651 repeated crystallization and sublimation possessed constant biological activity and a constant melting point af 166-167”. The analytical data agreed most closely with the empirical formula, CIIHI~O~N&. BIBLIOGRAPRY 1. Gyargy, P., Rose, C. S., Hofmann, K., Melville, D. B., and du Vigneaud, V., Science, 92, 609 (1940). 2. K6g1, F., and Tijnnis, B., 2. physiol. Chem., 242,43 (1936). 3. Allison, F. E., Hoover, S. R., and Burk, D., Science, 78,217 (1933). 4. West, P. M., and Wilson, P. W., Xcience, 89, 607 (1939). 5. Nilsson, R., Bjiilfve, G., and Burstrijm, D., Nuturwissenschuften, 27, 389 (1939). 6. GyGrgy, P., Melville, D. B., Burk, D., and du Vigneaud, V., Science, 91, 243 (1940). 7. du Vigneaud, V., Melville, D. B., Gyijrgy, P., and Rose, C. S., Science, 92, 62 (1940). 8. Gyijrgy, P., Kuhn, R., and Lederer, E., J. Biol. Chem., 131, 745 (1939). 9. Snell, E. E., Eakin, R. E., and Williams, R. J., J. Am. Chem. Sot., 62, 175 (1940). 10. Kiigl, F., Chem. and Ind., 67,49 (1938). Downloaded from www.jbc.org by on August 30, 2009