ARTICLE: THE REACTION OF FORMALDEHYDE WITH AMINO ACIDS Augustus Wadsworth and Mary C.

Pangborn
J. Biol. Chem. 1936, 116:423-436.

Access the most updated version of this article at http://www.jbc.org/content/116/1/423.citation Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites . Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/116/1/423.citation.full.h tml#ref-list-1

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

THE

REACTION

OF FORMALDEHYDE ACIDS
WADSWORTH AND MARY New and Research, Health, Albany) July

WITH

AMINO

BY AUGUSTUS (From the Division

C. PANGBORN York State

of Laboratories

Department

of
(Received

for publication,

6, 1936)

The studies here recorded form part of an investigation of the reactions that occur when diphtheria toxin is converted into toxoid and may also prove of value in further studies of the action of toxin in the tissues. When formaldehyde is added to toxin, it reacts with the various amino compounds present in the mixture. While the reaction between amino acids and formaldehyde has been extensively studied, the experimental conditions employed have usually been different from those which prevail in the formation of toxoid from toxin. The usual procedure at present for the preparation of diphtheria toxoid is to treat the toxin with from 0.3 to 0.4 per cent formalin (equivalent to 0.12 to 0.16 per cent of formaldehyde) at 37-39 for 4 weeks or more, the initial reaction being in the range of pH 7.6 to 8.4. The optimum concentration of formaldehyde varies with the medium in which the toxin is produced and appears to be related to the amino nitrogen content of the crude toxin; it is less than the amount theoretically equivalent to the amino nitrogen present. It is obvious that the results of experiments at high temperature, high pH, or high concentrations of formaldehyde have only an indirect bearing on the toxin problem.
A few investigators have reported studies of the reaction under conditions more or less resembling those of toxoid formation. Holden and Freeman (1) incubated several amino acids with 0.5 per cent formaldehyde in 0.1 N sodium hydroxide at 37 for 36 days. They found no reaction in acid solution, while in 0.1 N alkali there was a rapid initial reaction, followed by a further slow decrease in amino nitrogen. They also found that the simple amino acids were markedly less reactive than certain proteose and peptone preparations previously studied by Freeman (2). Gubareff 423

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

424

Formaldehyde

Reaction

with Amino Acids

and Bystrenin (3) investigated the effect of varying initial alkalinity and concentration of formaldehyde on the speed and completeness of the reaction with glycine at 38. Levy (4) studied the titration curves of a number of amino acids at 30 and concluded that the amino group may be associated with either 1 or 2 molecules of formaldehyde, but the complex containing 2 molecules apparently forms only in the presence of a large excess of formaldehyde. Tomiyama (5) determined equilibrium constants for the reaction of formaldehyde with glycine, alanine, and proline at 25 in a pH range from 8.0 to 10.0 and found that a constant was obtained only on the assumption that the combining ratio was 1. Both Levy and Tomiyama consider that their data indicate the formation of molecular compounds.

The purpose of the present study was to obtain data on the behavior of amino compounds of known constitution when treated with formaldehyde under conditions similar to those used for the production of diphtheria toxoid. The following substances were studied: (a) amino acids-glycine, alanine, lysine, cysteine, aspartic and glutamic acids, histidine, tryptophane, and arginine; (b) guanidine; (c) two synthetic dipeptides-alanylglycine and glycylalanine; and (d) peptone and a crude diphtheria toxin.
EXPERIMENTAL

The amino acids and guanidine were purchased from the Eastman Kodak Company. The peptone used was Difco proteose. The toxin was a crude diphtheria toxin produced in infusion-free medium prepared with the same peptone (6). Alanylglycine and glycylalanine were synthesized and purified according to the methods of Fischer (7).
Methods

The substancesto be studied were dissolved in 0.05 M phosphate buffer and the hydrogen ion concentration was adjusted to the desired level by the addition of 1 N sodium hydroxide. The pH range employed was 7.8 to 8.4 (determined calorimetrically). In most cases the concentration of the test substance was 0.05 M. Formaldehyde was added in an amount approximately equivalent to the amino nitrogen present. The solution was then made up to volume, mixed, and analyzed as soon as possible. When the reaction was very rapid, as in the case of cysteine, the initial values were determined by the analysis of control solutions. Toluene

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

A. Wadsworth

and M. C. Pangborn

425

was added as a preservative, and the solutions were incubated in paraffin-sealed bottles at 39. At intervals (usually after 2 and 24 hours, 7, 14, 21, and 28 days) samples were withdrawn and analyzed for (u) amino nitrogen by the Van Slyke microprocedure, (b) free formaldehyde, and (c) reversibly bound formaldehyde. Control solutions both of the amino compounds and of formaldehyde were found to be stable on incubation, except in the case of cysteine, which is gradually oxidized. Free Formaldehyde-The method was essentially that recommended by Velluz (8) and depends on the fact that the precipitation of formaldehyde by dimethyldihydroresorcinol (methone) can be made quantitative by the choice of appropriate conditions. As our method differed in some details from that of Velluz, it will be described here. The quantities used were adapted to micro and semimicro weighing. The sample to be analyzed, containing from 0.4 to 2 mg. of formaldehyde, was mixed with 20 cc. of a saturated aqueous solution of methone (4.5 gm. per liter). If necessary, 2 or 3 drops of 10 per cent acetic acid were also added to bring the final pH of the mixture in the range 4.4 to 5.0, since it was found that in the presence of amino compounds the pH must be 5.2 or lower to insure quantitative precipitation of free formaldehyde. After the mixture had stood at room temperature for 4 hours, the precipitate was collected on a microfilter, washed with water, dried for 2 hours at llO, and weighed. 1 mg. of formaldimethone is equivalent to 0.1027 mg. of formaldehyde. Warm acetone was used to wash the precipitate from the filter after weighing. When the solutions contained protein or peptone, it was found convenient to dissolve the wet precipitate from the filter with warm acetone, remove the acetone in a water bath, resuspend the purified precipitate in water, then filter, dry, and weigh. In agreement with Velluz, we found the method to be accurate to about 3 per cent. Reversibly Bound Formaldehyde--In order to study the reversal of the reaction between formaldehyde and amino compounds, the formaldehyde determination was modified. The solution to be analyzed was mixed with methone and the mixture was kept at 39 for 3 days before it was filtered and the precipitate weighed. Usually the amount of formaldimethone thus found was greater

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

426

Formaldehyde

Reaction

with Amino Acids

than that found in the free formaldehyde determination, indicating that exposure to methone at 39 had caused an appreciable splitting of the formaldehyde-amino compounds which had formed. The 3 day period does not necessarily give the maximum reversal possible with this reagent, since in a few cases longer periods of standing at 39 gave slight further increases in the amount of formaldimethone precipitated. For comparative purposes, however, it was considered more important to adopt a constant standardized procedure than to attempt a determination of the maximum reversal It was found in some control experiments with solutions of glycine and peptone which had reacted with formaldehyde that acidification of the reaction mixture to pH 4.8 with subsequent incubation at 39 was insufficient to reverse the reaction in the absence of methone. Results The experimental data are given in Tables I and II. It will be seen that the compounds studied varied greatly in the speed and completeness of their reaction with formaldehyde. The differences were, in general, in the same order as those found by Holden and Freeman, but were somewhat more marked at the higher hydrogen ion concentration employed by us. On continued incubation of the amino compounds with formaldehyde there was a steady decrease in the percentage of the total combined formaldehyde which could be split off by methone. There were four exceptions: (a) the two dipeptides, with which no reversal of the reaction was detected at any test period; and (b) arginine and guanidine, with which there was no significant change in the percentage of reversibly bound formaldehyde during the whole time of incubation. Table III shows the variations in reversibly bound formaldehyde, calcuIated from the data of Table I. From the gradual change in stability towards methone, it appears most probable that in these cases the reaction proceeds in two stages. The initial reaction may be quite rapid and is rather easily reversible. In the second stage, characterized by increased stability toward methone, the initial reaction product is probably transformed into a more stable compound by a secondary reaction or possibly a rearrangement.

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

A. Wadsworth

and M. C. Pangborn

427

These two &ages are further illustrated by the special cases of histidine and tryptophane. Figs. 1 and 2 represent graphically the data for histidine and tryptophane which are given/ in Table I; the curves for reversibly bound formaldehyde are omitted. The apparent combining ratio (moles of HCHO to moles of NHZ) varies strikingly and reaches the value of 1 only as the reaction approaches completion. It can scarcely be supposed that the true combining ratio varies in such a way, but it is quite reasonable to assume that the initial reaction product is more stable toward methone than toward nitrous acid (tryptophane) or vice versa (histidine), and that this initial product is gradually transformed into a second compound which is stable toward both reagents. In this case the apparent combining ratio would vary in the manner found by experiment. The reaction product of tryptophane and formaldehyde crystallized from the solution and was identified as 3,4,5,6-tetrahydro-4carbolined-carbonic acid. This substance was synthesized by *Jacobs and Craig by the condensation of tryptophane and formaldehyde in acid solution (10). When our product was compared with a sample of the carboline acid obtained from Dr. Jacobs, the two substances melted simultaneously with decomposition at 306 (uncorrected) and there was no depression of the melting point when the two were mixed. The yield of the carboline compound under the conditions of our experiment was nearly quantitative. The fact that this condensation takes place so readily under physiological conditions is of considerable interest, especially in view of the results of Hahn and his coworkers (11) who have prepared numerous 4-carboline derivatives from tryptamine at physiological temperatures in the pH range of 3.4 to 6.2. We are much indebted to Dr. Jacobs for the opportunity to compare the two compounds. Holden and Freeman state that formolized histidine also crystallized, but under the conditions of our experiments the product of the reaction between histidine and formaldehyde did not separate from solution. in Alkaline Solutions-The experiments on the Equilibria reversal of the reaction by methone were all carried out in acid solution (pH 4.4 to 5.0), since this acidity was necessary for the condensation of methone with formaldehyde. In slightly alkaline

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

TABLE

Analytical
2 hrs. [nitial Final --~24 hrs. ldays idays 11 day; 3 I 4 days 17 day) 11 da YS 28 days

Results

o.f Reactivity

of Amino

Compounds

with

Formaldehyde

(Mg.

per. 100 Cc :. 0.f Solution)

7-

Inihl

,
10.9 243 31.0 70.8 37.4 69.8 40.: 34.: 147.c 102.0 114.0 61.c 1 119.c 1 137.c I 105.0 112.0 8.2 42.6 35.0 147.0 34 .7 65 .3 14.7 31.0 58.2 8.0 4.8

-_

Cysteine*

37.3 82.8

Arginine

11.2 24.2 71.5 48.2 52.0 144.0 8.2 8.4

7.6

Guanidine

70.7 138.0 148.0 125.0

8.0 7.8

Glycine

72.0 144.0

55.0 76.0 148.0 107.0 116.0 67.4 136.0 144.0 65.7 129.4 141 .o 66.9 133.0 139.7 54.5 118.0 118.0 131.0 132.0 140.0 140.0 165.0 125.0 115.0 139.0 23.0 50.0 47.2 60.: 112.c 129.c 6O.t 111.c 123.c

Alanine

71.0 143.7 70.5 148.0 147.0

8.4

7.8

Alanylglycine

70.6 150.0

8.0

7.0

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

.O 103.0 .O 117.0 56.0 108.0 122.0 57.0 105.0 110.0 22 .O 19.2 27.2 25.7 60 .6 98.7 44.8 69.4
-

Glycylalanine

153.0

8.2 97 .O 35 .9 53 .O 95.0 28.7 44.7 8.0

8.2 7.0

Lysine

Amino Free HCHO Amino Free HCHO Free HCHO Amino Free HCHO Amino Free HCHO Amino Free HCHO Free HCHO Amino Free HCHO 32.5 66.c 68.0 95.8 95.8 107.0 69.5 97.0

N HCHO 39 N HCHO 39 HCHO 39 N HCHO 39 N HCHO 39 N HCHO 39 HCHO 39 N HCHO 39

140.0 142.0 165.0

Aspartic Glutamic Peptone 7.8 8.0 7.8 .P 8.4

acid1 $ (2%)

Amino I I

N <L

61.5 48.7 50.4 132.0 38.6 105 .o 118.0 53.7 84.0 117.0 -2 hrs. 4 days -14 hrs. -0.0 4 hrs. 5 hrs. 8 hrs. -13 hrs. 10 min. 1 hr. --

7.6 7.8 7.4

Toxin

Free HCHO HCHO 39 Amino N Free HCHO HCHO 39

68.5 115.0

61.5 48.5 29.2 71.6 88.0 46.5 54.2 93.0 ~-~~ 25.8 55.7 75.8 40.0 36.5 76.5

61.9 48.4 22.2 51.5 66.4 37.2 27.5 77.0

61.8 47.8 22.2 45.9 62.9 39.5 33.8 65.7

61.0 46.6 20.9 41.0 57.7 40.4 28.6 58.0

60.2 45.4 20.0 37.8 50.1 38.0 24.5 47.8

Histidine 15.5 15.6 2o.c 15.1 18.( 8.f 6.! 8.(

56.0 132.0 37.9 41.8 49.0 32.0 37.4 62.6 62.3 30.0 25.4 29.3 11.8 12.1 6.5 6.0 5.5

13.0 119.0

8.2 94.5 95.5

2.4 33.5 32.5

8.2

7.6

E Q 1.7 Trace 8.0

Tryptophane

Amino N Free HCHO HCHO 39 Amino N Free HCHO HCHO 39

42.5 85.0

g s

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

The line HCHO 39 gives the total amount of HCHO found after incubating the sample with methone for 3 days as described in the text. * At this concentration of cysteine the reaction had reached equilibrium in less than 15 minutes. The irregular results obtained later may be partly due to the instability of the acid. t Glycylalanine, like glycylglycine (9), gives abnormal results in the Van Slyke analysis; hence, the amino nitrogen figures are not reported. In this experiment the pH at first fell rapidly and was readjusted to 8.2 after 4 hours. 3 These two experiments were carried out at 36-37 instead of 39.

is CD

420

Formaldehyde
Percentage

Reaction
TABLE

with Amino Acids

II Reacting
-

of Components -

- -

e e

e E ss 00

c 4 -e

5 J : -

A 4 $;

Histidine Tryptophane Cysteine* Arginine Guanidine (0.045 Glycine Alanine Alanylglycine Glycylalanine (0.05 M)? Lysine (0.05 Aspartic Glutamic Peptone Toxin

Amino HCHO Amino HCHO Amino HCHO Amino HCHO 4

M)

N N N N

Ml i2 ?9 0 1 1 !3 L8

91 85 96 93 100 70 71 32 40 65 76 14 10 10 6 7.c 23 21 14

63

5:

61 5E 6:

if 77
18 1E 15 If l( 2: 1: 2: 6; 6,I 3: 23 58 0 0 56 61 46 76 3( 7: ( <: 5f 6: 4: 71 li 1,
l

83 2 -2:g g I?.-mole per 1. 0.040 0.044 0.03 0.029 0.025 0.028 0.05 0.05 0.042 0.05 0.048 0.05 0.048 0.05 0.05 0.05

Amino HCHO Amino HCHO Amino HCHO I

M)

N N N

6.4 5.f

0 0

54 56

2: 2i 2( , 2, GE 7: 8: 6( 31 7E 1 4 5E 6E 41 7: 3: 8: << ,

acidf 1 (2%)

Amino HCHO Amino HCHO Amino HCHO

N N * N

0 15

!3 81 !2 !7

11 30 0 0 42 46 32 53

49 58 42 68

0.10 0.055 0.043 0.036 6; 0.037 71 0.044 4 0.049 7! 0.038

* At this concentration of cysteine the reaction had reached equilibrium in less than 15 minutes. The irregular results obtained later may be partly due to the instability of the acid. 1 Glycylalanine, like glycylglycine (9), gives abnormal results in the Van Slyke analysis; hence, the amino nitrogen figures are not reported. In this experiment the pH at first fell rapidly and was readjusted to 8.2 after 4 hours. J These two experiments were carried out at 3637 instead of 39.

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

A. Wadsworth

and M. C. Pangborn

431

solution it was found that methone not only did not cause any reversal but did not prevent the continued condensation of formaldehyde with amino groups. It could not be assumed that the stability relationships revealed by the methone reaction necessarily corresponded to those existing in alkaline solutions because of the marked effect of changes in hydrogen ion concentration on the reaction between amino groups and formaldehyde. If the relaTABLE Percentage of Reversibly
E c N

III Bound
-

Formaldehyde

- --

Cysteine Glycine......... Alanine. Lysine. Peptone.. . Toxin. . Argininef. Guanidine Alanylglycine. Glycylalanine

4 2 ;s -81

k 4 d 85 63
1

k c 4 4 rz - .
77 75 28 19 LOO 52 0
-

5 2 71

-7

3 B -z _ -N
64 53 37

c 4 -I
53 39 13 9 13 26 97 0

100 100 48 100 100

47 27 26 64 51 96 100 50 0 0

20 20 39 97

13 18 34 47

0 -

The reversibly bound formaldehyde is the difference between the amount found on treating with methone for 3 days at 39 and that found in 4 hours at room temperature; this is recorded as a percentage of the total amount of formaldehyde which had reacted (initial value minus free formaldehyde value). This table represents the same series of experiments as Tables I and II. * Average of three experiments. t Average of two experiments.

tionships in alkaline solution can be predicted from the results of the methone reaction, it is evident that, in a mixture containing insufficient formaldehyde to react with all the amino groups, alanylglycine, for example, will gradually remove formaldehyde from its compounds with such acids as cysteine and arginine, and this will result in a decrease of reversibly bound formaldehyde in the mixture. This may serve as an example of the complex equilibria in a mixture of formaldehyde with crude toxin.

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

432

Formaldehyde

Reaction

with Amino Acids

0.1 M solutions of cysteine and of arginine were prepared in phosphate buffer at pH 8.2 and allowed to react with equivalent quantities of formaldehyde for 24 hours at 39. The solutions were then readjusted to pH 8.2. One portion of each solution was

120

100 . : E 4 40 60 80

I .o
I.8

01 i5
2 E .
0

1.6

$ I:

I.4

20

b.2

-.

--Q---m-

---____

--_

IO TIME

12

14 IN

16 HOURS

I8

20

22

24

-----

FREE FREE APPARENT

AMINO HCHO

N COMBINING RATIO of histidine with MOLS HCHO/MOLS NH2

FIG.

1. Reaction

formaldehyde

mixed buffer, buffer at 39

with and and and

an equal volume of 0.1 M alanylglycine in phosphate the remainder was diluted to 0.05 M with phosphate All solutions were incubated incubated as a control. analyzed at intervals as previously described. The

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

A. Wadsworth

and M. C. Pangborn

433

results are summarized in Table IV. It will be seen that there was a marked decrease in reversibly bound formaldehyde in the mixtures containing alanylglycine as compared with the controls containing cysteine or arginine alone. We may safely assume,

too . 8
E % 80

-1

60

g
40

20

IO TIME

12 IN

14

I6 HOURS

I8

20

22

24

-----

FREE FREE APPARENT

AMINO HCHO

N COMBINING RATIO MOLS with HCHO/MOLS NH2

FIG. 2. Reaction

of tryptophane

formaldehyde

then, that the behavior of the different formaldehyde-amino compounds toward methone shows their relative stability in alkaline solutions. It is interesting to compare this series of experiments with one reported by Holden and Freeman (l), who

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

434

Formaldehyde

Reaction

with Amino Acids

of or

were able to bring about a partial reversal of the combination formaldehyde with a metaprotein by incubating with histidine lysine.
DISCUSSION

It is evident that the analytical methods used in the present study are not capable of giving a complete picture of the reactions taking place. In considering the experiments on histidine and tryptophane, we were forced to conclude that the first products formed were unstable under the conditions of analysis. Obviously
TABLE

IV Solutions
Reversibly bound HCHO (mg. per 100 cc.)

Equilibria

in

Alkaline

jl a 3 .j B Alanylglycine 0.05 M -k arginine 0.05 M.. Arginine 0.05 M. Alanylglycine 0.05 M + cysteine 0.05 M....................... Cysteine 0.05 M.. 8.2 8.2 8.2 8.2 7.4 7.6 8.2 8.2

0 cd 12 :z$ 2 b b x -~-_ 3 .~-2 --~--

Time

after

mixing

05 OS + j $ $ N 07 d

86.376 176 38.925.620.417.6 I 86.: 83 92.6 92.696 96 90 92.494 101 97 82 89 64.941.923.813.4 73 65.560.556.5

* In this case the initial value is that found immediately after mixing the solutions of the two amino acids, after the cysteine and arginine solutions had reacted with formaldehyde for 24 hours.

it is possible that the same may be true at least in the initial stages of the other experiments. This does not invalidate the use of the data for comparative purposes so long as it is emphasized that the terms free amino nitrogen and free formaldehyde are used in a relative rather than an absolute sense and are defined by standardized conditions of analysis. The acids which show the greatest affinity for formaldehydehistidine, tryptophane, arginine, cysteine (and, in the experiments of Holden and Freeman, tyrosine)-are those which have other functional groups in the molecule in addition to the amino and carboxyl groups. The second carboxyl group in aspartic

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

A. Wadsworth

and M. C. Pangborn

435

and glutamic acids decreases the reactivity. The simple amino acids, alanine and glycine, are markedly less reactive than the two dipeptides prepared from them. In the case of arginine, the analytical data indicate 2 moles of formaldehyde reacting in 0.05 M solution for each mole of arginine. Since guanidine itself combined with formaldehyde under similar conditions, it seems probable that the guanidine group of arginine also reacts with formaldehyde, although Sorensen (12) found that this group could not be determined by the form01 titration method. With regard to the mechanism of the reaction, it may be recalled that Hahn and his coworkers (11) found evidence of two stages in the reaction between tryptamine and substituted pyruvic acids. They considered the first step to be the formation of an addition compound which then loses water to form the carboline ring. It seems highly probable that the mechanism of the reaction between tryptophane and formaldehyde is similar and that the unstable compound indicated by our data is the hydroxymethyl compound which splits off water, yielding the stable carboline acid. It is interesting also to compare our results with those of Tomiyama (5). A calculation from Tomiyamas equilibrium constant for the reaction between glycine and formaldehyde indicates that at the concentrations used by us nearly 50 per cent of the formaldehyde would be associated with glycine as a molecular compound at 25, while by chemical methods we find only about 10 per cent combined even after 4 days at 39. Evidently the two sets of data do not represent the same reaction; the molecular compound detected by physicochemical methods must be immediately dissociated when chemical methods of analysis are used. We suggest that, under the experimental conditions described by us and, therefore, also during the production of toxoid, there may be at least three stages in the combination of formaldehyde with amino groups: (a) a loosely associated molecular compound; (b) a labile chemical compound, possibly a hydroxymethyl or methylene derivative, indicated by the reversible stage in our experiments; and (c) a stable compound probably formed by further reaction or rearrangement of compound (b). In studies of toxoid production it is evidently insufficient to draw a distinction between free and bound formaldehyde, as the bound formaldehyde may be either reversibly or irreversibly combined,

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

436

Formaldehyde

Reaction with Amino Acids

while some of the free formaldehyde may be loosely associated with amino groups to form molecular compounds.
SUMMARY

1. Data are presented on the course of the reaction of formaldehyde with several amino acids and related compounds under conditions comparable to those in which diphtheria toxin is transformed into toxoid. 2. In most cases two stages in the reaction can be demonstrated: (a) a rapid, reversible reaction; (b) a slower, irreversible one. The reversal of the reaction has been studied in both acid and alkaline solutions.
BIBLIOGRAPHY

1. Holden, H. F., and Freeman, M., Australian J. Exp. Biol. and Med. SC., 8, 189 (1931). 2. Freeman, M., Australian J. Exp. Biol. and Med. SC., 7, 117 (1930). 3. Gubareff, E., and Bystrenin, A., Biochem. Z., 266, 92 (1932). 4. Levy, M., J. Biol. Chem., 99, 767 (1932-33). 5. Tomiyama, T., J. Biol. Chem., 111, 51 (1935). 6. Wadsworth, A., and Wheeler, M. W., J. Infect. Dis., 66, 123 (1934). 7. Fischer, E., Ann. Chem., 340, 123 (1905); Ber. them. Ges., 37, 2486 (1994). 8. Velluz, L., Compt. rend. Sot. biol., 111, 289 (1932). 9. Dunn, M. S., Butler, A. W., and Deakers, T., J. BioZ. Chem., 99, 217 (1932-33). 10. Jacobs, W. A., and Craig, L. C., J. BioZ. Chem., 113, 759 (1936). 11. Hahn, G., Blirwald, L., Schales, O., and Werner, H., Ann. Chem., 620, 107 (1935). 12. Siirensen, S. P. L., Biochem. Z., 7, 45 (1908).

Downloaded from http://www.jbc.org/ by guest on May 30, 2013

Downloaded from http://www.jbc.org/ by guest on May 30, 2013