PAPER CHROMATOGRAPHY OF VITAMIN Blz AND RELATED BACTERIAL GROWTH FACTORS*

BY WALTER A. WINSTEN AND EDWARD EIGEN

(From the Food Research Laboratories, Inc., Long Island City, New York]

(Received for publication, April 18, 1949)

In a preliminary note (l), the present authors reported that Lactobacillus Eeichmannii 313 is capable of using six alternative factors for growth in vitamin Bn-deficient media. Two of these were found in vitamin Blz concentrates and presumably represent two forms of the vitamin. Hoff- mann et al. (2) previously noted that crystalline antipernicious anemia factor (APA) (vitamin B1.J supports the growth of L. Zeichmannii 313. Of the other four factors, one was provisionally identified as thymidine, a substance which was reported by Snell et al. (3) as supporting the growth of this organism. The three remaining growth factors are of unknown composition.

In the present report, a paper chromatographic procedure for separating the alternative growth factors is described as well as the method of recog- nizing the positions of the several factors on a chromatogram by use of L. Zeichmannii 313 as a microbiological indicator. Extension of the method to the examination of other natural materials has revealed the existence of at least one other substitute growth factor replacing vitamin Bti in the nutrition of the test organism.

That the naturally occurring alternative growth factors which replace vitamin Bn in the nutrition of L. Zeichmannii 313 are desoxyribosides would appear probable from evidence presented in this paper. Kitay et al. (4) have shown that desoxyribosides can replace thymidine in the nutrition of several lactic acid bacteria.

The paper chromatographic technique has aided in the interpretation of assay values for apparent vitamin Bn activity as measured by the usual tube assay.

EXPERIMENTAL

Paper Chromatography and Development of Bioautographs-The general method involving the use of paper chromatography in the analysis of growth factors which occur in nature in more than one chemically defined form has been described by Winsten and Eigen (5). Briefly, what has

* Part of the material in this paper was presented at the Thirty-third annual meet- ing of the Federation of American Societies for Experimental Biology, Detroit, April 18, 1949.

109

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110 VITAMIN B12

been termed the bioautographic technique consists of subjecting a droplet of a solution containing the substitute growth factors to paper chro- matography. The resultant paper chromatogram is then laid on the sur- face of nutrient agar seeded with an appropriate bacterial culture. The nutrient agar contains all other factors necessary for the growth of the test organism, except any one of the complex of substances being studied. After removal of the paper strips and incubating, zones of microbial growth form at different positions along the locus of the strip chromatogram. The positions of the various substitute growth factors depend on their Rp values for the solvent system employed in developing a chromatogram and serve to characterize and identify the different factors present in the

TABLE I Vitamin BIZ Basal Medium (Double Strength) - ---- __- --

nn-Tryptophan n-Cystine Dr.-Alanine L-Asparagine Glutamine Adenine Guanine Uracil Xanthine Pyridoxal hydrochloride Thiamine “ Riboflavin Niacin

T 800 mg. 400 “ 400 “ 200 ‘(

40 ‘( 20 (‘ 20 “ 24 “ 20 “

500 Y 400 I‘ 400 “

1200 l‘

-

Biotin Pyridoxine hydrochloride Calcium pantothenate Folic acid p-Aminobenzoic acid Casein hydrolysate*

“ digestt Dextrose Sodium acetate K,HPO, Tween 80 Salts B$ Adjust to pH 6.8 and add d

to make 1 liter

0.8~ 2400 “ 800 ‘I

1000 “ 20 “

100 ml. 25 l1 40 gm. 40 I‘

5.0 “ 2.0 “

10 ml. tilled water

* Acid-hydrolyzed casein obtained from the Nutritional Biochemicals Corpora- tion, Cleveland, Ohio.

t Enzymatic digest of casein obtained from Nutritional Biochemicals. $‘ MgSOI.7Hz0, 10 gm.; NaCl, 0.5 gm.; FeS0,.7Ha0, 0.5 gm.; MnS01+4H20, 0.5

gm.; dissolved in 250 ml. of distilled water.

original sample. The incubated plate which reveals the developed chro- matogram by this direct microbiological procedure has been termed a bioautograph.

In the present study on vitamin Bl2 and substitute growth factors, 10 to 30 ~1. samples of the solutions being examined (pH 5.0) were spotted on Whatman No. 1 paper strips. The chromatograms were developed overnight at room temperature with wet n-butanol in the apparatus described by Winsten (6). The strips were allowed to dry in air for 1 hour at 30-35”. They were then laid on agar plates seeded with L. leichmannii 313. The composition of the double strength basal medium is described in Table I. The medium was diluted a-fold, and 1.5 per cent agar was

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W. A. WINSTEN AND E. EIQEN 111

added. The medium is fundamentally similar to that suggested by Hoff- mann et al. (2).

The organism L. Leichmannii 313 was maintained on Difco tryptose agar stabs prepared weekly. The daily transplants were made in a broth containing 2 per cent tryptose and 2 per cent litmus milk. The cultures mere incubated at 37”.

For use on the agar plates, a 24 hour culture of the organism in the tryptose-milk broth was transplanted to 10 ml. of single strength basal medium to which 2 mpgm. of vitamin B= were added. After 24 hours, the culture was centrifuged for 15 minutes at 2500 R.P.M. and was resus- pended in 10 ml. of physiological saline. The culture was again centrifuged and suspended in 10 ml. of saline. The resultant suspension was then added to 300 ml. of melted basal medium maintained at a temperature of 42-50”. The seeded agar was poured on rectangular plates 9.5 X 16 inches.

After laying the dried paper strip chromatograms on the hardened agar and allowing the moist agar to leach the strips for 5 minutes, they were removed and the plate was incubated overnight at 37”. The resulting zones of bacterial growth were always quite light and, in order to facilitate observation, it was necessary to hold the plate at an angle with a source of light to one side and behind it. When so observed, the zones were always sharply defined, well formed ellipses. The edges were outlined in the agar with a sharp instrument. Contact photographic prints were then prepared with Kodagraph contact standard paper. The zones were finally marked in ink on the contact paper, making a permanent record.

Samples of the various materials analyzed contained 0.1 to 10.0 y per ml. calculated as vitamin BE, determined by a titrimetric tube assay procedure with L. leichmannii 313.

On occasion, samples were first subjected to the proteolytic action of protease 15, an enzyme preparation manufactured by Rohm and Haas. Usually 5 ml. of a suspension (or solution) of material calculated to give a final solution containing the above range of apparent vitamin B1z ac- tivity were treated with 10 mg. of protease at pH 7.0 for 18 hours at 37” in the presence of toluene.

Grateful acknowledgment is made for the various materials analyzed, among which may be mentioned a vitamin Blz concentrate and a prepa- ration of crystalline vitamin Blz supplied by Dr. W. L. Sampson and Dr. W. H. Ott of Merck and Company; crystalline APA (in solution), a fer- mentation animal protein factor (APF) preparation, and a thymidine sample supplied by Dr. T. H. Jukes and Dr. A. L. Franklin of the Lederle Laboratories; an acid precipitate of the cow manure factor from Dr. H. R. Bird; and a sample of hypoxanthine desoxyriboside supplied by Dr. E. E. Snell. In addition, several commercial parenteral liver preparations for

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112 VITAMIN Big

use in the treatment of pernicious anemia were examined. Other materials tested included condensed fish solubles, a commercial stomach concen- trate intended for use in pernicious anemia, corn steep liquor, a commercial trypsin, Wilson’s liver powder 1: 20, and an enzyme digest of sperm desoxy- ribonucleic acid.

Results of Qualitative Bioautographic Analysis for Vitamin BU and Alterna- tive Growth Factor-Fig. 1 shows a schematic diagram of a typical set of

3

-

0

:_

0 0

2

0 0

3

8 0 0

4

0

0 Q 0

- 5

FIG. 1. Bioautogrsphs of preparations containing vitamin BE together with sub- stitute growth factors. Test organism, L. Zeichmannii 313. Chromatograms de- veloped for 18 hours at room temperature with wet n-butanol; 10 to 20 ~1. samples were chromatographed. Strip 1, crystalline vitamin B12 preparation, 3.8 y per ml.; Strip 2, parenteral liver Preparation 1,0.67 y per ml. of apparent vitamin Bncontent; Strip 3, corn steep liquor, about 0.25 y per gm. of apparent vitamin Bn, diluted 1:2; Strip 4, corn steep liquor, diluted 1:5; Strip 5, vitamin BIZ Concentrate 2,1.5 y per ml. of apparent vitamin Bn.

bioautographs obtained as described above. From an examination of Strip 2, which represents the bioautograph of parenteral liver Preparation 1, it will be noted that the slowest moving zone (at the top of the strip) has a double character, suggesting two substances. Analysis of crystalline vitamin Bn preparations and vitamin Ba concentrates indicates that the doublet zone of growth may be due to two forms of vitamin Ba. In this connection it is of interest to recall the two forms of APA factor reported by Smith (7).

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W. A. WINSTEN AND E. EIOEN 113

Further examination of Strip 2 reveals the presence in this preparation of three other substances which move more rapidly on the chromatogram. The nature of these factors will be discussed below.

It was found t,hat the RF value for a given growth factor present in sev- eral preparations was independent of the type of preparation examined within esperimcntal error. Thus on a given day the RF value of the most rapid factor was 0.55 for parenteral liver Preparation 1, 0.54 for parenteral liver Preparation 2, and 0.54 for parenteral liver Preparation 3. However, from day to day the RF value varied anywhere from 0.52 to 0.67. As a consequence, in identifying new factors on any given day a well studied material (the parenteral liver Preparation 1) was also analyzed. It was further found t,hat, if the most rapidly moving substance was assigned a value of 1.0 for its rat,e of movement, the rates of movement of the other substances relative to it were reasonably constant. This was true so long as the daily temperature variations were minor. Toward the end of the present study, with the onset of the higher summer laboratory temper- atures, the relat,ive rates changed somewhat in a non-uniform manner. This may be a consequence of the fact that the percentage change of Rp value with temperature was not the same for all the alternative growth factors.

In Table II are summarized the relative rates of movement of the various alternative gronth factors, the most, rapidly moving substance found in t.he thymidine preparation being assigned a rate of 1.0.

Included in the vitamin IL column of Table II are all factors which were so slow moving, when wet n-butanol was the developing solvent, that accurate Z& v&es could not be obtained. On occasion, only a single zone of growth was observed on a bioautograph at the site of application of the test sample.

It is evident that vitamin IL2 may be more complex in character than is indicated by the terms double and single zones of growth. Clearly a more efficient solvent than wet n-butanol is needed to resolve the substances that cause the characteristic double zone of growth associated with so called crystalline vitamin I& preparations. Experimental results obtained with such a solvent n-ill be described in a later publication.

It is of interest that all prepa,rations so far tested, which were intended for the treatment of pernicious anemia, contained a growth factor or factors which moved slowly or not at, all when n-butanol was used as a solvent. Alternative growth fact,ors of greater solubility in wet ?z-butanol relative to water were found in many of the cruder pernicious anemia preparations as well as in preparations of fermentation APF and condensed fish solubles.

In an investigation of corn steep liquor, one factor was found which did not fit in with those indicated in Table II. In Fig. 1 are shown bio- autographs for two dilutions of corn steep liquor (see Strips 3 and 4).

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114 VITAMIN BB

It is evident, on comparing these with the bioautograph for parenteral liver Preparation 1, that at least one of the two slowest moving growth factors in corn steep liquor must represent a fifth alternative growth factor in addition to the four of Table II. The average relative rates of the growth in corn steep liquor were 0.51, 0.63, 0.76, and 1.0. Inspection of

TABLE II Relative Rates of Movement of Substitute Growth Factors for L. Leichmannii dlS*

Preparation

Parenteral liver Preparation 1 “ “ ‘I 2 “ “ “ 3

Trypsin (l%, crude) Vitamin Bl* Concentrate 1

‘I I‘ “ 2 I‘ ‘I crystalline

Cow manure factor

Fermentation APF preparation Wilsonliver preparation, 1:20 (lOoJo) Stomach concentrate (enayme-

treated) Condensed fish solubles

Crystalline APA factor Thymidine

Growth factors present

Vitamin Blnt (type Jf zone of growth)

Double “ “

Double ‘I “

Single (no movement)

Double ‘I

Single (no movement)

Single (no movement)

Double

-

Average.........................................

Substitute factors

l I 2 I 3 14 Relative rates in terms of Factor 4

0.32

0.32

0.54 0.51 0.54 0.51

0.56

0.56

0.52

0.50

- 0.53

-

0.78 0.77 0.83

0.77

0.76 0.80 0.78

0.75$

0.78

- 1.0 1.0 1.0 1.0

1.0

1.0 1.0 1.0

1.0

1.0

1.0

* Chromatography on Whatman No. 1 paper overnight with wet n-butanol as the solvent.

t The two forms of vitamin Biz moved too slowly, preventing an accurate meas- urement of their relative rates. On occasion bioautographs exhibit a single zone in the region normally occupied by vitamin B 12 in place of the double most often observed.

# Very small zone.

the average values in Table II suggests that the factor in corn steep liquor with a relative rate of 0.63 represents the new substance. Thus, in addi- tion to at least two forms of vitamin Blz, five other substitute growth factors are present in various natural materials.

Evidence of Desoxyriboside Nature of Alternative Growth Factors-That the

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substitute growth factors dealt with in Table II represent desoxyribosides appeared probable, as mentioned earlier in this paper. To throw light on this question, we attempted to identify one of the factors with thymidine, the only desoxyriboside available to us at the start of this investigation. From Table II it is apparent that the thymidine preparation contained two factors. While it seemed probable that the most rapid factor (assigned relative rate, 1.0) which produced the larger zone of growth was thymidine, proof of this fact was lacking.

Since only a small amount of thymidine was available, the method of Hot&kiss (8) could not be applied. A second indirect method was available, however. By the use of Leuconostoc citrovorum 8081 in a bio- autographic method, an organism which utilizes thymidine in place of an uncharacterized factor, as described by Sauberlich and Baumann (9), it was found that the thymidine preparation caused a zone of growth at a position identical, within experimental error, with that of the faster, larger zone obtained when L. leichmannii 313 was the test organism.

The slower factor in the thymidine preparation (see Table II) was inactive for L. citrovorum 8081. These findings therefore serve to identify the factor assigned a relative rate of 1.0 as thymidine.

Subsequent bioautographic testing of hypoxanthine desoxyriboside indi- cated that the preparation, kindly made available by Dr. E. E. Snell, was free of any other factors and that this desoxyriboside moved with a relative rate, indicating its possible identity with Substitute Factor 2 in Table II.

Additional evidence suggesting the desoxyriboside nature of the more rapidly moving growth factors other than vitamin BE was afforded by bioautographic analysis of an enzyme digest of desoxyribonucleic acid. The digestion was carried out substantially in the manner described by Chargaff et al. (10) for the production of desoxyribonucleosides.

A sample of sperm desoxyribonucleic acid (DNA), obtained from the Nutritional Biochemicals Corporation, Cleveland, Ohio, was dissolved with the aid of a little alkali to give a 4 per cent solution. The pH was adjusted to 6.7. Bioautographic analysis of this solution of DNA re- vealed the presence of a vitamin Blz-like factor (i.e., it did not “move” on a paper chromatogram when n-butanol was the solvent).

To a 1 ml. sample of the DNA solution, 1 ml. of Verona1 buffer of pH 6 to 7, containing 2 per cent MgSOd*3HzO, and 1 ml. of an enzyme solution were added. The enzyme solution contained 300 y per ml. of crystalline des- oxyribonuclease (Worthington Biochemical Laboratory) and 50 mg. per ml. of mylase P (Wallerstein Laboratories). After incubation at about 30” overnight under toluene, bioautographic analysis showed the presence of four substitute growth factors in addition to the vitamin Bn-like factor

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116 VITAMIN Ba

originally present before enzyme digestion. A control analysis of the en- zyme solution indicated the substantial absence of such factors.

The relative rates of movement of the factors were 0.57, 0.69, 0.78, and 1.00. The relative rates of the substitute growth factors in parenteral liver Preparation 1, subjected to bioautographic analysis at the same time as the enzyme digest of DNA, mere 0.60, 0.79, and 1.00. The first figure (0.60) for liver Preparation I is higher than that indicated in Table II for the same liver preparation. As was pointed out earlier, this is probably a conse- quence of a temperature change in the laboratory. The data of Table II were obtained at about 25 ‘. The temperature in the present experiment was much higher (about 31’). If we take account of this change in relative rates, the factors found in the DNA digest are possibly hypoxanthine des- oxyriboside (relative rate 0.57) and thymidine (relative rate 1.0).

The other two factors (relative rat.es 0.69 and 0.78) may represent other desoxyribosides in the enzyme digest of DNA. For brevity, a diagram of the bioautograph of the DNA digest has not been given. The main factors present in the digest (i.e., t,he ones causing the largest zones of growth) were those with the relative rates 0.57 and 1.0. This finding may have a bearing on the relative rates of liberation of the desoxyribosides by enzyme diges- tion, and on their relative activity for L. leichmannii 313.

The four factors found in corn steep liquor may represent the same des- oxyribosides found in the DNA enzyme digest. Their occurrence in corn steep liquor may be a consequence of bacterial fermentation, since the corn steep liquor examined was highly ferment.ed.

The only factor which so far has not been implied to be a desoxyriboside is that one with a relative rate of 0.32 (see Table II). That it is such a substance seems probable.

Quantitative Analysis of Vitamin BE and Substitute Growth Factors in Parenteral Liver Preparatio+-It has been shown above that it is possible to separate vitamin B,, and various alternative growth factors by the use of paper chromatography. Assuming that all the growth factors which barely move away from the site of application of a test sample on a chromatogram (when wet n-butanol is used as a developing solvent) represent forms of vitamin B12, it is possible to assay such factors in various concentrates in a simple fashion. Such an assay was carried out on parenteral liver Prepara- tion 1 (see Fig. 1, Strip 2). In this assay, several 8.6 ~1. samples of the parenteral liver preparation were subjected to chromatographic separation on paper strips in the usual way. After being dried in air, each strip was cut in two at a point, just below the estimated position of the vitamin Blz entities (about 1.5 inches beneath the site of application of the test sample). The upper sections of the strip chromatograms therefore contained the slow moving vitamin Bi2. The lower sections contained the faster moving al- ternative growth factors.

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W. A. WINSTEN AND E. bt@Ei’t 117

The top and bottom sections were each eluted with a known volume of water and aliquots were taken for assay of total apparent vitamin Blz ac- tivity. In carrying out the assay, a 72 hour titrimetric tube assay was employed with the basal medium given in Table I.

In addition to the above, as controls, unchromatographed samples were also assayed. As a further check, instead of cutting some of the strip chromatograms after development, the entire strips were eluted and as- sayed. Finally, in order to demonstrate that there is no loss in vitamin Blz during chromatography, several samples of vitamin Blz Concentrate 1 were also chromatographed; the chromatograms were cut and the top and bot- tom sections were assayed for vitamin B1z. Every assay was carried out in

TABLE III Quantitative Assay of Fractions of Parenteral Liver Preparation I after Separation 0%

Paper Chromatoorams

Parenteral liver 0.64 0.67 Preparation l* 0.66 0.75

0.19 0.18 0.73 0.74

Vitamin Brt Con- 1.93 1.99 centrate 1 2.03

None - --

* In carrying out determinations on the cut sections of the chromatograms, two top sections and two bottom sections were eluted together; in all eight chromato- grams were sectioned and combined in this way for the four determinations.

Not chromatographed 0.67 Top of chromatogram 0.73 Bottom of chromatogram 0.19 Entire chromatogram eluted 0.76 Not chromatographed 2.08 Top of chromatogram 2.00 Bottom of chromatogram None

Deter- Deter- mina- mina-

tion tion 2 3

-__

0.70 0.67 0.80 0.80 0.17 0.17 0.73 0.73 2.00 1.93 2.08 2.08

None None

Apparent activity calculated as crystalline vitamin Bn, y per ml.

- Material analyzed IDeter-

mina- tion

1

Drter- mina- tion

4 .verage

quadruplicate with crystalline vitamin Blz as the standard. The results appear in Table III.

It will be noted that after chromtiography there was somewhat more apparent vitamin Blz activity per ml. at the top of the chromatogram com- pared to the value obtained without chromatography. The bottom section of the chromatogram, containing as it did the three fast moving alternative growth factors, contributed an additional amount of apparent vitamin Blz activity, which was only about 25 per cent of the activity at the top of the chromatogram.

It would appear, in the present instance at least, that, in a total tube assay of an unchromatographed sample, the value obtained (0.67 y per ml.) was not the sum of the activities contributed by the top and bottom sets of factors measured separately. This finding raises a question concerning

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1’18 VITAMIN B~.J

the joint growth-promoting effects of vitamin Bi:! and thymidine. While several authors have noted that these substances can replace one another in supporting the growth of certain lactic acid bacteria, there is little de- tailed information on the quantitative aspects of their combined action. Lack of sufficient pure t,hymidine has prevented us from obt,aining pertinent data on this problem.

From Table III, it is also plain that little or no vitamin Blz is lost during the development of the paper chromatograms, since all of the vitamin applied as Concentrate 1 was recovered by eluting t,he top sections of the chromatograms.

In Fig. 1, consideration of the size of the zones of growth in the bioauto- graph for the parenteral liver Preparation 1 (see Strip 2) would lead one to believe that most of the activity would have been found in the bottom sec- tions of the chromatograms of Table III. The small area of the double zone of growth caused by the very slow moving vitamin B,z is probably due to poor diffusion in the agar because of its high molecular weight.

Note-Stokstad et al. (11) have presented evidence that, when vitamin Ble is autoclaved with a basal medium, there is some destruction of the vitamin. The amount of destruction varies with the preparation being analyzed. These authors pointed out that inclusion of thioglycolic acid (TGA) in the medium protected vitamin BD from destruction by autoclaving. In the light of these observations, it was of interest to repeat the experiment, the results of which were described in Table III. The vitamin Blz content of an unchromatographed sample of parenteral liver Preparation 1, determined in the presence of TGA during autoclaving, was 0.60 y per ml., about the same as is indicated in Table III. (Other parenteral preparations exhibited anywhere from 50 to 100 per cent more vitamin Blz activity when TGA was present during autoclaving, confirming the findings of Stokstad et cd.) Quadruplicate assay of the top of a chromatogram, in the presence of TGA, yielded a value of 0.80 y per ml., again in agreement with the value of Table III. Assay of the bottom of a chromatogram in the presence of TGA yielded 0.02 y per ml., only about 10 per cent of the figure noted in Table III. Thus the apparent vitamin Blz activity of faster moving alternative growth factors was much less when measured in the presence of TGA.

DISCUSSION

The application of the bioautographic method to the study of vitamin BLz and substitute growth factors for L. leichmannii 313 has revealed marked similarities among such diverse materials as parenteral liver prep- arations intended for the treatment of pernicious anemia, a fermenta- tion APF preparation, and condensed fish solubles. Fish solubles have been used by many authors as sources of APF for chicks. While it has

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W. A. WINSTEN AND E. EIGEN 119

been implied that the APF activity of fish solubles may be due to the vitamin PW content, of the preparation, no evidence other than the growth- promoting effect for chicks and perhaps for lactic acid bacteria has been presented. In the present paper, t)he finding in condensed fish solubles of one factor that does not (‘move” on a paper chromatogram under the conditions described, which is thus similar to authentic vitamin B12, has been demonstrated. In addition there are present two other more rap- idly moving growth factors, found on occasion in a wide variety of other sources of vitamin B12.

As mentioned earlier, the question has yet to be answered as to whether the more mobile growth factors for L. leichmannii 313, which are probably desoxyribosides, have APF or antianemia activity. The more mobile fac- tors by themselves display apparent vitamin Blz activity in preparations, such as corn steep liquor, which lack the vitamin itself as evidenced by the absence of any “immobile” growth factor. This finding suggests that all studies on the apparent vitamin B,z content of natural materials determined in the usual form of tube assay should be checked by a bioautographic study to ascertain the types of growth factors present.

It has recently been reported by Nichol et al. (12) that one can obtain liver preparations which are active in the treatment of pernicious anemia but possess no animal protein factor activity for the chick. It would be of great interest to determine what growth factors, if any, for L. leichmannii 313 are present in such preparations.

SUMMARY

1. A paper chromatographic study of vitamin Blz and related growth fac- tors has been carried out. In addition to vitamin B,a, five other substitute growth factors for Lactobacillus leichmannii 313 have been recognized. Evidence of the desoxyriboside nature of the substitute growth factors has been presented.

2. A quantitative analysis for vitamin Br2 which comprises the joint use of paper chromatography and a tube assay of the separated factors has been described.

BIBLIOGRAPHY

1. Winsten, W. A., and Eigen, E., J. Biol. Chem., 177,989 (1949). 2. Hoffmann, C. E., Stokstad, E. L. R., Franklin, A. L., and Jukes, T. H., J. Biol.

Chem., 176, 1465 (1948). 3. Snell, E. E., Kitay, E., and McNutt, W. S., J. Biol. Chem., 176, 473 (1948). 4. Kitay, E., McNutt, W. S., and Snell, E. E., J. Biol. Chem., 177, 993 (1949). 5. Winsten, W. A., and Eigen, E., Proc. Sot. Exp. Biol. and Med., 67, 513 (1948). 6. Winsten, W. A., Science, 107, 605 (1948).

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120 VITAMIN Ba

7. Smith, E. L., Nature, 161, 638 (1948). 8. Hotchkiss, R. D., J. Biol. Chem., 1’76, 315 (1948). 9. Sauberlich, H. E., and Baumann, C. A., J. Biol. Chem., 176, 165 (1948).

10. Chargaff, E., V&her, E., Doniger, R., Green, C., and Misani, F., J. Biol. Chem., 177,405 (1949).

11. Stokstad, E. L. R., Dornbush, A. C., Franklin, A. L., Hoffmann, C. E.,Hutchings, B. L., and Jukes, T. N., Federation hoc., 8, 257 (1949).

12. Nichol, C. A., Robblee, A. R., Cravens, W. W., and Elvehjem, C. A., J. Biol. Chem., 177, 631 (1949).

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Walter A. Winsten and Edward EigenBACTERIAL GROWTH FACTORS

AND RELATED12VITAMIN BPAPER CHROMATOGRAPHY OF

1949, 181:109-120.J. Biol. Chem. 

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