V O L U M E 19, NO. 4, A P R I L 1 9 4 7
277
the operations of filtering and washing required from 8 to 10 minutes, and the volumes of the washings ranged from 47 to 59 ml. Titration. If the amount of sulfur is believed to be not much in excess of 5 mg., add 5 to 10 ml. of water, close the flask tightly with a rubber stopper, and shake vigorously to disintegrate the filter paper. Remove the stopper and wash it and the neck of the flask with a stream of hot water from a wash bottle. Introduce 2 drops of 0.5y0 phenolphthalein indicator and, manipulating the flask by means of a holder, titrate with 0.03 N sodium hydroxide, with vigorous agitation and occasional heating, until the end point is obviously near. If the amount of sulfur is much greater than 5 mg. the initial addition of water should be omitted, and the disintegration of the paper deferred until about 10 ml. of the alkali have been added; this procedure avoids an unduly large volume of liquid a t the end point. Just short of the end point, heat the rmxture to active boiling, and then titrate to an unmisfakable and moderately deep pink color which survives a final boiling of several seconds. The titration may be completed by whole or half drops, but the end point is not sufficiently sharp to warrant attempted further rehement. I n the trials the volumes a t the end of the titrations ranged from 25 to 32 ml. Blank Analysis. Transfer to a 150-ml. Erlenmeyer flask 100 ml. of water. As appropriate add 5 ml. of 3% hydrogen peroxide or 1.5 ml. of bromine water, and then 0.1 or 0.25 gram of hydroxylamine hydrochloride. Introduce 10 ml. of 2y0 benzidine hydrochloride reagent, and proceed thereafter as in the analysis, titrating finally to the same end color. The color identity must be judged from memory, as the color does not persist long enough to permit matching. The blanks obtained in the trials were uniformly close to 0.2 ml. of 0.03 N sodium hydroxide. It is advisable to conduct also an over-all blank which includes decomposition of some sucrose or benzoic acid in the bomb. I n the trials this did not increase the blank obtained as above. Test Analyses of Organic Compounds. Table I1 presents the results of analyses of a number of organic sulfur compounds by the procedure given above. There are included also results obtained when no auxiliary oxidant was used and, for several compounds, results obtained gravimetrically (barium sulfate), to confirm the accuracy of the volumetric benzidine method. It will be noted that analyses of a number of compounds in absence of an auxiliary oxidant gave results which are acceptable
and which in some cases were not improved by use of hydrogen peroxide or bromine water. The need for an auxiliary oxidant is perhaps to be presumed when sulfur is present in unoxidized condition, but it seems advisable as a general procedure always to include one of the supplementary oxidizing agents tested. The report (7, cf. 16)that sulfur compounds oxidizable to sulfonic acids may not be properly decomposed in the oxygen bomb was not confirmed in the cases that might test this claim, both benzyl disulfide and ethyl benzenesulfonate giving acceptable results. A single analysis requires about 2.5 hours. By use of two sample cups and by suitable arrangement of the work six analyses can be completed, and a seventh sample decomposed, in 8.5 hours. ACKNOWLEDGMENT
Grateful acknowledgment is made to the Faculty Research Committee of the University of Pennsylvania for grants to aid this study. Preliminary studies by Doris Koch Cavalieri were useful in the development of the procedure. LITERATURE CITED
(1) Alicino, IND.ENQ.CHEM.,ANAL.ED.,13, 506 (1941);Am. 800. Teating Materials, D271-43, p. 13. (2) Bradley, Corbin, and Floyd, Zlzd. Ens. C h . , 18, 583 (1926). (3) Cnllen and Toenniea, IND.ENG.CHIIM., ANAL.ED., 13, 469 (1941). (4) Clauder, Magyar Gyogysreresrtud Tarsmag Erteoitdje, 11, 246 (1935). (5) Fiske, J. Biol. Chem., 47,59 (1921). (6) Friedheim and Nydegger, 2. angcw. C h m . , 20, 9 (1907). (7) Griffin, IND.ENQ.CHEM.,ANAL.ED.,1, 167 (1929). (8) Haese, 2 . angew. C h m . , 40,595 (1927). (9) JBrvinen, Ann. acad. aci. Fcnnicac, 2, N o . 4, 22; Chem. Zentr., 192, I, p. 526. (10) Lincoln, Carney, and Wagner, IND.ENQ.CHEM.,ANAL.ED.,13, 358 (1941). (11) Meldrum and Newlin Zbid., 1, 231 (1929). (12) Miiller and Dtirkea, Bar., 35, 1587 (1902). (13) Owen, Biochem. J.,30, 352 (1936). (14) Skau and Newell, IND.ENQ.CHEM.,ANAL.ED.,5, 180 (1933). (15) Weisselberg, Petroleum Z . , 30, N o . 33, 1 (1934).
VITAMIN B, GROUP Determination Extraction Procedures for the Microbiological of Vitamin B, d
JESSE C. RABINOWITZ AND EShlOND E. SNELL Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wis.
S
I S C E much of the vitamin B6 present in natural materials is not available to microorganisms ( I ) , the determination of this factor by microbiological assay requires preliminary treatment of the sample to liberate the vitamin Be. Atkin et al. (1) have suggested for this purpose either hydrolysis of a sample containing 2 to 4 micrograms of vitamin Bs with 180 cc. of 0.055 N sulfuric acid for 1 hour at 1.4 kg. per sq. cm. (20 pounds per sq. in.) pressure, or enzymatic digestion with clarase. Other investigators have hydrolyzed with 1 or 2 N sulfuric or hydrochloric acid (IO,19). Melnick et al. (6) showed, however, that the vitamin B6 content of a dried yeast sample hydrolyzed with 2 N sulfuric acid was much lower than the value obtained when 0.055 N acid was used for the hydrolysis. They ascribed this t o destruction of an acid-labile substance possessing vitamin Bo activity. This investigation waa originally undertaken to compare the effectiveness of various procedures for liberating vitamin Bs
from natural materials, preliminary to a study of the relative distribution of pyridoxal, pyridoxamine, and pyridoxine in natural materials, and to determine, if possible, whether an additional, acid-labile form of vitamin Bs, as reported by Melnick el al. (4, exists. In the meantime, a preliminary note by Rubin, and Scheiner (6) reported that, although hydrolysis with 2 N sulfuric acid yields lower values than hydrolysis with 0.055 N acid or clarase, this does not result from destruction of vitamin B, by the 2 N acid, since subsequent treatment of the 2 N acid hydrolyzates with clarase liberates as much vitamin Bs as the original clarase treatment. Some of this unusual behavior may result from the presence of pyridoxal phosphate in natural materials. The growthpromoting properties of this substance for microorganisms have not been reported previously. The action of various hydrolytic procedures on a sample of this synthetic coenzyme has been studied and compared with results of the same procedures a p
A N A L Y T I C A L CHEMISTRY
218
Pyridoxal phosphate is less than 10% as active as an equimolar amount of pyridoxal in promoting growth of yeast and various lactic acid bacteria. Hydrochloric acid, 0.055 N,hydrolyzes the coenzyme more rapidly than stronger acid under otherwise similar conditions. With Saccharomyces carlsbergensis 4228, which responds equally to the three known forms of vitamin Be, as the assay organism, various hydrolytic procedures for the release of vitamin Be from natural materials were compared. A procedure for the extraction of vitamin Be which is applicable to most natural materials is recommended.
plied to natural products. On the basis of these experiments, a modification of an existing procedure for extraction of vitamin Be has been developed which is superior to those now in use, and which appears to release all of the vitamin Be in natural materials in a form available to yeast. EXPERIMENTAL
Assay Methods. Vitamin Be was determined with the yeast Saccharomyces carlsbergensis 4228, essentially as described by Atkin et al. ( I ) , but with minor modifications. These modifications, which resulted in decreased growth in the “blank” tubes and an increased response to added vitamin Be, were as follows: Of the solutions, only the casein hydrolyzate was prepared differently than has been suggested (1). S.M.A. Corp. “vitaminfree casein’’ was hydrolyzed and decolorized with charcoal as described elsewhere (12). Assays were carried out on one half the scale recommended in the original method. Samples, with water to make the volume to 2.5 cc., were steamed for 10 minutes in Pyrex tubes (25 X 200 mm.). The basal medium was steamed in a separate container and, when cooled, inoculated with 0.01 mg. of moist yeast per 2.5 cc. Then, 2.5 cc. of the inoculated basal medium were added aseptically to each tube. This inoculum is about l / , ~that recommended by Atkin et .ul. ( 1 ) . Great care was taken to avoid exposure of samples and assay tubes to light because of its destructive effect on vitamin B8 (2). The tubes were mechanically shaken for 18 hours at 30” C., then steamed for 5 minutes t o stop growth of the yeast. Two cubic centimeters of water were added to each tube before reading in the Evelyn colorimeter, which was set to read 6-cc. volumes. In a typical assay the transmission of tubes containing 0, 2, 5, 10, 15, 20, and 25 millimicrograms of pyridoxine hydrochloride was 99, 88, 76, 60, 47, 38, and 30y0 of the incident light, respect ively. Growth-Promoting Activity of Pyridoxal Phosphate. The growth-promoting activity for several microorganisms of a sample of synthetic pyridoxal barium phosphate, kindly supplied by I. C. Gunsalus, was determined with the results shown in Table I. Determinations with Lactobacillus cbsei, Streptococcus fuecalzs, and Leuconostoc mesenteroides were made as described previously (1.2). The latter culture was No. 9135 of the American
Table I. Comparative Activities of Pyridoxal and Pyridoxal Phosphate for Various Organisms“ Leuco. mesenYeast leroides b L. casei S. faecalis Hydrolysis P ridoxal HPz,ridox& &idox;p&10, Procedure H A PO4 None 1.0 0.01 1.0 0.03 1.0 0.02 1.0 0.03 Clarase 0 1.1 0.34 0.9 0.29 1.1 0.25 ... ... 0.1NHCld 1.1 0.34 0 . 9 0.26 0.9 0.27 1.0 0.27 1 NNaOHd 1.0 0.01 . . , 0.04 . 0.03 . . . .. , a Sample of synthetic pyridoxal barium phosphate shown t o contain 32 to 36% pyridoxal as determined spectrophotometrically or by Scudi phenol tests (9). b Organism grown on medium used for S. faecalis as described by b e l l and Rannefeld (7). C 1 mg. of clarase was incubated with 1007 of pyridoxal barium phosphate or pyridoxal hydrochloride for 24 hours a t 37’ C. in a 0.1 M solution of sodium acetate at p H 4.5. d Autoclaved 60 minutes a t 15 poqnds’ pressure
HPyidoxaa,
..
Type Culture Collection, which, in contrast to Leuconostoc mesenteroides P-60, required vitamin Bc for growth. This sample had been shown to contain 32 to 36% pyridoxal as determined spectrophotometrically or by the Scudi phenol test ( 3 ) . The unhydrolyzed coenzyme was only 3 to 10% as active as the hydrolyzed coenzyme for any of the microorganisms on which it was tested. Clarase digestion or hydrolysis with 0.1 Ai hydrochloric acid released the expected amount of the vitamin for 5’. curlsbergensis; slightly smaller amounts were indicated by assay with the other test organisms. Alkaline hydrolysis was ineffective.
TIME IN HOURS
Figure 1. Liberation of Pyridoxal from Pyridoxal Phosphate by Acid at 15 Pounds’ Pressure
The rate of release of pyridoxal from the coenzyme by acid hydrolysis was further investigated. Fifty-microgram samples of pyridoxal barium phosphate were added to 10 cc. of either 0.055 S or 2 S hydrochloric acid and autoclaved 1 kg. per sq. cm. (15 pounds per square inch) pressure for varying lengths of time. Hydrolysis with 0.055 S hydrochloric acid effected rapid and complete release of pyridoxal, whereas 2 S hydrochloric acid hydrolyzed the coenzyme much more slowly (Figure 1). Vitamin Be Content of Natural Materials. To determine the optimal conditions for release of vitamin Be from natural materials, amounts of the sample estimated to contain about 2 micrograms of vitamin & were hydrolyzed as follows:
( a ) . With 180 cc. of 0.055 N hydrochloric acid for time intervals varying from 1 t o 12 hours at either 15 or 20 pounds’ steam pressure per square inch. ( b ) . With 10 cc. of 2 N hydrochloric acid as in (a). [When 2 N acid is used, the same high ratio of acid to sample used with 0.055 N acid cannot be employed, since the sodium chloride formed on neutralizing the acid extract preliminary to assay becomes inhibitory t o the assay organism. The highest concentration of sodium chloride which can be tolerated without partial inhibition varies somewhat from one assay to another, but is approximately 50 mg. per assay tube (5 cc. of diluted medium).]
279
V O L U M E 19, NO. 4, A P R I L 1 9 4 7
tion of maximum amounts of vitamin B6 for all samples tested except Vitab (a rice bran concentrate). For dried yeasts, liver powders, and plant products, 5 hours’ hydrolysis was significantly more effective in the liberation of vitamin than hydrolysis for 1 hour. With two fresh meats, however, very little difference was observed between the two treatments. Hydrolysis of dried yeasts, liver powders, and fresh meats with 2 N hydrochloric acid for 1 hour gave low values. However, if this procedure waa extended to 5 hours, the values obtained a p proached, in most cmes, the maximum values observed after hydrolysis with 0.055 N acid. The plant products showed somewhat different and more irregular results. In general, hydrolysis for 1 hour with 2 N hydrochloric acid released maximum or very nearly maximum amounts of 5 the vitamin. Continued hydrolysis for 5 hours I I I I I I resulted in only slight further changes. Atkin 2 4 6 8 IO 12 et al. ( 1 ) specifically recommended use of 0.44 N TIME IN HOURS in place of 0.055 N sulfuric acid for release Figure 2. Liberation of Vitamin B6 from a Dried Yeast by Hydrolytic of vitamin Bs from cereal products. The presProcedures ent authors have not investigated use of this concentration of acid for this DurDose. . - and Following hydrolysis, all samples were cooled, adjusted to pH believe it should be satisfactory. 5.0, diluted to 250 cc., filtered, and assayed. Clarase digestion gave maximal values for vitamin Bs in samples of dry yeast; however, low values were found by this These hydrolytic procedures differ from those investigated procedure for liver powders and plant products. by Atkin et al. (I) in that hydrochloric acid was substituted for contents of these mateWith few exceptions, the vitamin sulfuric acid and the time of autoclaving was varied to a greater rials as determined by rat assay were somewhat lower than the extent. Limited comparative trials showed no difference in the values obtained by microbiological assay. effectiveness of these two acids. This result rould be expected in view of the recent demonstration by Rubin et al. ( 7 ) that comDISCUSSION pleteness of extraction of vitamin BP,from yeast by sulfuric acid was dependent upon the pH of the extraction mixture. The superiority of low concentrations of acid over higher conTKe vitamin content of samples containing more than 10 centrations in releasing pyridoxal from pyridoxal phosphate is micrograms of vitamin Bg per gram of material was also deterunusual, but has also been observed by Gunsalus and eo-workers mined after digestion with clarase. (4). The fact that vitamin Bg is released more rapidly from the coenzyme than from natural samples when treated with 0.055 N For this purpose, an amount of sample which contained about hydrochloric acid probably indicates that in the latter process 2 micrograms of vitamin B, and an equal weight of clarase were suspended in 10 cc. of a 1 M solution of sodium acetate. The pH more is involved than hydrolysis of the phosphate linkage in was adjusted to 5.0 and 2 cc. of benzene mere added. After inpyridoxal phosphate. cubation for 24 hours at 37” the sample was diluted to 250 cc., filtered, and assayed. The vitamin Bg content of clarase, 3.3 micrograms per gram, was subtracted from the final value found. I
The results obtained by acid hydrolysis of a dried yeast sample (R.C. S o . 6, supplied by Dr. Atkin), are shoxm in Figure 2. The maximum values for vitamin Bg were obtained for this yeasf, after hydrolysis at 20 pounds’ pressure for 3 to 5 hours with 0.055 A’ hydrochloric acid or at 15 pounds’ pressure for 9 to 12 hours with 0.055 A’ hydrochloric acid. Hydrolysis of the yeast with 2 A’ hydrochloric acid at 15 or 20 pounds’ pressure resulted in the much slower release of vitamin Ba. However, after 12 hours’ hydrolysis with the 2 N acid, the amount of vitamin Bg found was only 12% lower than the maximum value obtained by weak mid hydrolysis. The use of 20 pounds’ pressure effected more rapid release of vitamin Be than did the use of 15 pounds’ pressure n ith the corresponding concentration of acid. This yeast sample is not unique in its behavior under these conditions of hydrolysis. The rate of release of vitamins B6 from a sample of Wilson’s 1 to 20 liver powder is shown in Figure 3. Under these conditions, the maximum value for vitamin B6 was also obtained only after 5 hours’ hydrolysis. The results of applying the various hydrolytic treatments to a variety of products is shown in Table 11. The values shown in the last column were obtained in an independent investigation (8) by assay of the same products with rats. Hydrolysis with 0.055 iV hydrochloric acid for 5 hours resulted in the libera-
L
TIME
IN HOURS
Figure 3. Liberation of Vitamin BOfrom Wilson’s 1 to 20 Liver Powder by Hydrolysis with 0.055 N Hydrochloric Acid a t 20 Pounds’ Pressure
280
ANALYTICAL CHEMISTRY
Table 11. Vitamin Bc C o n t e n t of Natural Materials after Hydrolytic Treatment” Substance
Dried veasts Atkih’syeastb 2 6 . 5 Rubin’syeastc 2 4 . 0 Yeast, G-AB 20.7 Wilson liver powdtz s Fraction L 11.7 1:2Od
Whole substanoe Fresh meats Beef kidney Lamb leg Plant roducte Vitab Cerophyl Soybean flour Dried oats Whole wheat flour Whole yellow corn Dry green peasd
Hydrolytic Procedure 0.065 N HC1 1 hour 6 hours Chase Micrograms per gram
2 N HC1 1 hour 6 hours
Rat Assay
38.0 33.9 25.1
36.2 31.8 24.1
44.5 37.4 29.6
40.4 40.3 27.1
24.7
42.0
18.3 56.5
16.5 47.6
21.4 61.2
11.2 34.8
14.6 33.0
11.6
12.7
13.3
14.2
8.4
10.0
8.6 3.2
8.1 3.3
9.8 4.3
9.9 4.6
.... ....
10.2 4.5
83.8 8.2 7.6 1.7
83.6 8.7 6.9 1.6
30.0 8.9 6.1 1.4
67.4 11.2 8.0 1.9
64.5 8.4
51.7
4.4
4.1
2.3
4.1
4.6
3.4
5.4
5.7
1.9
2.0
1.2
1.9
.... .... ....
.... ....
39.5
.. . .
6.6
the vitamin Be content of most products (Table 11) is to be expected on the basis of the observations that a major portion of the vitamin Bs of some tissues consists of pyridoxamine and pyridoxal (If), and that these compounds are 1-8 active than is pyridoxine when fed to rats with the ration ( 8 ) ,but are equally 88 active as pyridoxine for the yeast, Saccharomyces carlsbergensis. The higher values obtained by rat assay for some products low in vitamin B e . g . , rolled oats, Table 11-may result from intestinal synthesis of vitamin Bg in the rat when the high proportions of these products necessary for assay are added to the ration. That such intestinal synthesis can occur is shown by the resulta obtained when dextrin is fed in the deficient ration for rats (8). An alternative explanation, of course, would be that complete release of vitamin Bs is not effected in all products by the procedures investigated here.
7.1 2.6 2.6 4.3
4.0
Determined with Saccharomyces carlsberpensis. Activities of pyridoxine hydrochloride, pyridoxal hydrochloride, and pyridoxamine dihydroohloride were 1.0, 1.1, and 0.81, respectively. b Sample of dried yeast R.C., No. 6 , kindly supplied by L. Atkin. * Sam le of dried yeast 200 B , kindly supplied by S. Rubin. pre&usly desoribet(6). d Samples used for rat and microbiological assay were not the same in thwe three cases. a
In attempting to explain the superiority of low concentrations over higher concentrations of mineral acid in releasing vitamin Bg from most natural materials, Rubin et al. ( 7 ) showed that optimal release occurred between pH 1.5 and 2.0, with a maximum at pH 1.7 to 1.8. On either side of this pH range, release of vitamin Be *as markedly less efficient. From the limited data available, pyridoxal appears to conetitute a minor fraction of the total vitamin Be present in hydrolyzed extracts of most natural materials, including yeast ( 6 , I f ) . This point requires further investigation with improved hydrolytic procedures, but if true, hydrolysis of pyridoxal phosphate cannot explain increases in total vitamin Be of the magnitude noted following hydrolysis with low concentrations of mineral acids. I t is probable that a phosphate of pyridoxamine (cf. 14) also occurs naturally, the hydrolysis of which contributes to the observed increase. That a bound form of pyridoxine of unknown nature occurs in rice bran concentrates is well known (6, 11). This conjugate appears not to be present in yeast or animal tissues, and furthermore is hydrolyzed only by relatively high concentrations of acid ( 5 ) . Its possible presence would not, therefore, explain increases of the magnitude observed with animal tissue and yeast following hydrolysis with 0.055 N hydrochloric acid. Prolonged hydrolysis of all samples tested with 2 N hydrochloric acid either had no significant effect or resulted in an increase in the amount of vitamin Bg available to yeast. Destruction of vitamin B, under these conditions of hydrolysis was not observed. This result lends no support to the hypothesis (6) that an acid-labile form of vitamin Be occurs naturally. I t should be emphasized, however, that the postulation of an acidlabile forin of vitamin B, was based on more than the apparent destruction of activity for S. carhbcrgensis by hydrolysis with strong acid (6). It was also observed that assay with S. faecalis, which responds to only two of the three known forms of vitamin Be, gave higher values for the vitamin Bc content of yeast than did =say with S. carlsbergensis, which responds equally to all three, and that the factor responsible for this increased activity for S. faecalis was largely destroyed by acid hydrolysis. Thus the present results, and those of Rubin et al. ( 6 , 7 ),do not disprove the existence of such a labile component of the vitamin Bg group. That lower values were obtained with rats than with yea& for
SUMMARY
Pyridoxal phosphate is much less active than an equimolar amount of pyridoxal in promoting growth of yeast, Laetobacillw casei, Streptococcus faecalis, and Leuconostoc mesenteroides. Autoclaving with 0.055 N hydrochloric acid for 1 hour at 15 pounds’ pressure effected completed hydrolysis of the coenzyme; higher concentrations of acid were much less effective. Various hydrolytic procedures for the release of vitamin Be from natural materials were compared. Previously suggested procedures for this purpose were found inadequate for general we. The most effective single procedure was to autoclave with 0.055 N hydrochloric acid a t 20 pounds’ pressure for 5 hours. This procedure gave maximal values for all products tested except a rice bran concentrate, and is suggested as the method of preference for general use. Rice bran concentrate required hydrolysis with 2 N acid for maximal release of vitamin Be. This, procedure wag also ad* quate for some other plant products, but was markedly inferior to treatment with 0.055 N acid for yeast and animal products. Although, with the latter products, vitamin B, was released much slower by the 2 N acid than by 0.055 N acid, no evidence for destruction of vitamin Ba by the stronger acid was obtained. Except for some cereal grains, which are low in vitamin Be, values obtained by yeast assay are as high or somewhat higher than those obtained with rats. The reasons for this discrepancy are discussed briefly. LITERATURE CITED
Atkin, L., Schultz, A. S.,Williams, S. L., and Frey, C. N., IND. ENG.CHEM., ANAL.ED.,15, 141 (1943). (2) Cunningham, E., and Snell, E. E., J . Biol. Cham., 158, 491 (1)
(1945).
Gunsalus, I. C., Umbreit, W. W., Bellamy, W. D., and Foust. C. E., Ibid., 161,743 (1945). (4) Gunsalus, I. C., and co-workers, private communication. (5) Melnick, D., Hochberg, M., Himes, H. W., and Oser, B. L.,
(3)
J . Biol. Chem., 160, 1 (1945).
Rubin, S.H., and Scheiner, J., Ibid., 162,389 (1946). Rubin, S. H., Scheiner, J., Drekter, L., and Hirshberg, E., Abstracts, 110th Meeting, AMERICANCHEMICALSOCIHITY, Chicago, Ill. (September 1946). (8) Sarma, P. S., Snell, E. E., and Elvehjem, C. A., J . Biol. C h m . , (6) (7)
165, 55 (1946).
Sarma, P. S.,Snell, E. E., and Elvehjem, C. A., unpublished data. (10) Siegel, L., Mehick, D., and Oser, B. L., J . Biol. Chem., 149,361 (9)
(1943). (11) (12) (13)
Snell, E. E., Ibid., 157,491 (1945). Snell, E. E., and Rannefeld, A. N., Ibid., 157,475 (1945). Stokes. J. L.. Larsen. 8..Woodward, W. R., and Foster, J. W..
(14)
Umbreit, W, W., O’Kane,D. J., and Gunsalus, I. C., J . Bact., 51,
~I
Zbid., 150,17 (1943). 576 (1946). PRESESTED in part before the Division of Biological Chemistry at the 110th Meeting of the AMERICAN CHEMICAL SOCIETY, Chicago, Ill. Supported in part by a grant from the Wisconsin Alumni Research Foundation. Published with the approval of the director of the Wisconsin Agricultural Erperiment Station. For the preceding paper, No. IX of this seriea, see Sarms. Snell, and Elvehjem, Proc. SOC.E z p t l . Biol. Med., 63, 284 (1946).
