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Biotin Vitamin

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Biotin Vitamin
ISOLATION OF BIOTIN (VITAMIN H) FROM LIVER*

BY VINCENT DU VIGNEAUD, KLAUS HOFMANN,t AND

DONALD B. MELVILLE1

(From the Depurtment of Biochemistry, Cornell University Medical College,

New York City)









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AND PAUL GYORGY

(From the Babies and Childrens Hospital, and the Department of Pediatrics,

School of Medicine, Western Reserve University, Cleveland)

(Received for publication, June 6, 1941)



In the course of our work on the isolation of vitamin H, the anti-

egg 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, experi-

mentally and from the literature, on distribution, stability, solu-

bility, 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,









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injected for 30 days, necessary to bring about complete disappear-

ance 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 phos-

photungstic acid, followed by decomposition of the precipitate

with barium hydroxide (8). The vitamin H activity of the result-

ing 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 demon-

strated 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-

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 chromato-

graphic 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









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with dilute HCl, and the extract was then evaporated to dryness.









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” (uncor-

rected). 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 crystal-

lized, 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 communica-

tions (6, 7). Expressed in terms of vitamin H units, the various

preparations of purified product that we have made have all con-

sistently 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









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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 com-

pound, as shown in the experimental part, agree most closely with

the empirical formula of CnH,,03N$. Molecular weight deter-

minations by the Rast method with various solvents were not

entirely satisfactory. However, all the results obtained with bor-

neol 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 deriva-

tives 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, how-

cvcr, in solubility, in crystalline form, and in composition leads

us to believe that the compound isolated by us from liver is iden-

tical 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

du Vigneaud, Hofmann, Melville, Gyiirgy 647



for yeast growth and for anti-egg white injury activities.1 How-

ever, 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









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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 Saccharo-

myces cerevisiae was used as the test organism and turbidity meas-

urements 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 tur-

bidity readings observed are a linear function of the yeast growth.

Each sample to be assayed was made up in a series of decimal dilu-

tions 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. Com-

parison of the interpolated concentration at which half maximum

growth occurred in the assay sample with the corresponding con-

centration obtained in the same manner with a solution of known

vitamin H activity permitted calculation of the vitamin H equiv-

alence of the sample being assayed.

Isolation Procedure, EsteriJication-500 cc. of the vitamin H

concentrate from liver mentioned in the introduction were con-

centrated 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 meth-

anol 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 bicar-

bonate, and the mixture was shaken until all of the bicarbonate









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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 adsorp-

tion. 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 col-

lected 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 vita-

min 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 con-

centrate 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 solu-

tion 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

du Vigneaud, Hofmann, Melville, Gyijrgy 649

TABLE I

Distribution of Biotin Activity in First Chromatograph

Vitamin

Weight Activity H units

Column elated with Fraction No.

Zli3’



cc. w7. vitaminH units

Chloroform 300 339-x1 15,000 147,000 9.8

Acetone 300 339-VIII 805 1,000,OOO 1242

“ 90% + meth- 50 339-1x 21 45,000 2140

an01 10%

“ ‘I 50 339-x 76 145,000 1910









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iI “ 50 339-x1 256 310,000 1210

‘I ‘I 50 339-x11 102 45,000 440



Total ._..........................._..., 16,260 1,692,OOO





TABLE II

Distribution of Biotin Activity in Second Chromatograph

-







Column &ted with I kaction No. Weight Activity





cc. ml. Zamin H unit B



Acetone 200 343-I 622 l,ooo 1.6

“ 90% + meth- 130 343-11 583 Inactive 0

anol 10%

“ “ 700

10 343-111 82 8.5

‘I ‘I 1110

10 343-IV 72 80,000

I‘ ‘I 10 343-v 52 178,000 3420

“ I< 10 45 162,000

343-VI 3600

“ L‘ 10 343-VII 42 220,000 5240

“ ‘I 10 343-VIII 37 220,000 5950

“ “ 200,000

10 343-1X 30 6670

‘I “ 10 343-x 89 312,000 3500

‘I LL 10 343-X1 65 240,000 3700

CL L‘ 162,000

10 343-X11 68 2380

I‘ “ 10 343-X111 76 120,000 1580

6‘ “ 10 343-XIV 50 60,000 1200

-.



Total 1913 1,955,700





to 343-X111 were combined and used for the next step. Frac-

tions 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 potas-

sium bicarbonate was then added to the solution until it was alka-

line to litmus and the solution was extracted with three 50 cc.









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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 crystal-

lizations from a mixture of methanol and ether yielded a material

that melted at 166-167”. The material was sublimed at 140-

150’=’ in a high vacuum (10v6 mm.) and recrystallized from meth-

anol 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 fol-

lowing composition.



CJMhN~S.

Calculated. C 51.14, H 7.02, N 10.84, S 12.42, OCH3 12.0

Found. “ 51.40, “ 7.19, “ 10.84, “ 12.16, “ 11.5



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 (vita-

min H) from liver has been described. The compound after

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 for-

mula, 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).









Downloaded from www.jbc.org by on August 30, 2009

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).


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