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J. Biol. Chem. 1936, 116:423-436.
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It is obvious that the results of experiments at high temperature. While the reaction between amino acids and formaldehyde has been extensively studied. Health.4 per cent formalin (equivalent to 0.jbc. 2013 . or high concentrations of formaldehyde have only an indirect bearing on the toxin problem.4. the initial reaction being in the range of pH 7.org/ by guest on May 30. the experimental conditions employed have usually been different from those which prevail in the formation of toxoid from toxin.1 N sodium hydroxide at 37” for 36 days.1 N alkali there was a rapid initial reaction. Albany) July WITH AMINO BY AUGUSTUS (From the Division C. Holden and Freeman (1) incubated several amino acids with 0.THE REACTION OF FORMALDEHYDE ACIDS WADSWORTH AND MARY New and Research. followed by a further slow decrease in amino nitrogen. high pH. while in 0.12 to 0. They also found that the simple amino acids were markedly less reactive than certain proteose and peptone preparations previously studied by Freeman (2). They found no reaction in acid solution. it reacts with the various amino compounds present in the mixture. it is less than the amount theoretically equivalent to the amino nitrogen present.16 per cent of formaldehyde) at 37-39” for 4 weeks or more. 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. A few investigators have reported studies of the reaction under conditions more or less resembling those of toxoid formation. The usual procedure at present for the preparation of diphtheria toxoid is to treat the toxin with from 0.3 to 0. When formaldehyde is added to toxin.6 to 8. 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. 6. PANGBORN York State of Laboratories Department of (Received for publication. Gubareff 423 Downloaded from http://www.5 per cent formaldehyde in 0.
The peptone used was Difco proteose.4 (determined calorimetrically). and arginine. In most cases the concentration of the test substance was 0.424 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”. as in the case of cysteine.8 to 8. Both Levy and Tomiyama consider that their data indicate the formation of molecular compounds. alanine. When the reaction was very rapid. (b) guanidine. lysine. The toxin was a crude diphtheria toxin produced in infusion-free medium prepared with the same peptone (6). Methods The substancesto be studied were dissolved in 0.0 to 10. alanine. The pH range employed was 7. Toluene Downloaded from http://www. and proline at 25” in a pH range from 8. The solution was then made up to volume. Formaldehyde was added in an amount approximately equivalent to the amino nitrogen present. Alanylglycine and glycylalanine were synthesized and purified according to the methods of Fischer (7).jbc. and (d) peptone and a crude diphtheria toxin. EXPERIMENTAL The amino acids and guanidine were purchased from the Eastman Kodak Company. mixed.05 M. tryptophane.org/ by guest on May 30. cysteine. The following substances were studied: (a) amino acids-glycine.0 and found that a constant was obtained only on the assumption that the combining ratio was 1. but the complex containing 2 molecules apparently forms only in the presence of a large excess of formaldehyde.05 M phosphate buffer and the hydrogen ion concentration was adjusted to the desired level by the addition of 1 N sodium hydroxide. 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. histidine. 2013 . Tomiyama (5) determined equilibrium constants for the reaction of formaldehyde with glycine. (c) two synthetic dipeptides-alanylglycine and glycylalanine. 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. the initial values were determined by the analysis of control solutions. aspartic and glutamic acids. and analyzed as soon as possible.
dried for 2 hours at llO”.1027 mg. and 28 days) samples were withdrawn and analyzed for (u) amino nitrogen by the Van Slyke microprocedure.5 gm. If necessary. 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. remove the acetone in a water bath. then filter. per liter).jbc. C. and (c) reversibly bound formaldehyde. containing from 0. As our method differed in some details from that of Velluz. except in the case of cysteine.4 to 5.org/ by guest on May 30. dry.0. When the solutions contained protein or peptone. 1 mg. At intervals (usually after 2 and 24 hours. The quantities used were adapted to micro and semimicro weighing. Reversibly Bound Formaldehyde--In order to study the reversal of the reaction between formaldehyde and amino compounds. we found the method to be accurate to about 3 per cent. of a saturated aqueous solution of methone (4. washed with water. since it was found that in the presence of amino compounds the pH must be 5. Pangborn 425 was added as a preservative. 14. of formaldehyde. it will be described here. In agreement with Velluz. The sample to be analyzed. of formaldimethone is equivalent to 0.2 or lower to insure quantitative precipitation of free formaldehyde. resuspend the purified precipitate in water. of formaldehyde.A. the formaldehyde determination was modified. it was found convenient to dissolve the wet precipitate from the filter with warm acetone. 21. 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. was mixed with 20 cc. which is gradually oxidized. (b) free formaldehyde. Control solutions both of the amino compounds and of formaldehyde were found to be stable on incubation. and the solutions were incubated in paraffin-sealed bottles at 39”. 7. Warm acetone was used to wash the precipitate from the filter after weighing. After the mixture had stood at room temperature for 4 hours. and weigh. and weighed. Wadsworth and M. 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. 2013 . Usually the amount of formaldimethone thus found was greater Downloaded from http://www. the precipitate was collected on a microfilter.4 to 2 mg.
indicating that exposure to methone at 39” had caused an appreciable splitting of the formaldehyde-amino compounds which had formed. in general. but were somewhat more marked at the higher hydrogen ion concentration employed by us. Table III shows the variations in reversibly bound formaldehyde. 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. The 3 day period does not necessarily give the maximum reversal possible with this reagent. In the second stage. characterized by increased stability toward methone. since in a few ‘cases longer periods of standing at 39” gave slight further increases in the amount of formaldimethone precipitated. From the gradual change in stability towards methone. The initial reaction may be quite rapid and is rather easily reversible. calcuIated from the data of Table I. it appears most probable that in these cases the reaction proceeds in two stages. There were four exceptions: (a) the two dipeptides. It will be seen that the compounds studied varied greatly in the speed and completeness of their reaction with formaldehyde. For comparative purposes.jbc. the initial reaction product is probably transformed into a more stable compound by a secondary reaction or possibly a rearrangement. 2013 . with which there was no significant change in the percentage of reversibly bound formaldehyde during the whole time of incubation.org/ by guest on May 30.426 Formaldehyde Reaction with Amino Acids than that found in the free formaldehyde determination. and (b) arginine and guanidine. Results The experimental data are given in Tables I and II. with which no reversal of the reaction was detected at any test period. however. in the same order as those found by Holden and Freeman. 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.8 with subsequent incubation at 39” was insufficient to reverse the reaction in the absence of methone. The differences were. Downloaded from http://www.
When our product was compared with a sample of the carboline acid obtained from Dr. the two substances melted simultaneously with decomposition at 306’ (uncorrected) and there was no depression of the melting point when the two were mixed. Figs. The yield of the carboline compound under the conditions of our experiment was nearly quantitative. It can scarcely be supposed t’hat the true combining ratio varies in such a way.4 to 5.0). We are much indebted to Dr. Pangborn 427 These two &ages are further illustrated by the special cases of histidine and tryptophane. 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.A. C.jbc. and that this initial product is gradually transformed into a second compound which is stable toward both reagents. The reaction product of tryptophane and formaldehyde crystallized from the solution and was identified as 3. The fact that this condensation takes place so readily under physiological conditions is of considerable interest. 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). In this case the apparent combining ratio would vary in the manner found by experiment.6-tetrahydro-4carbolined-carbonic acid. Holden and Freeman state that formolized histidine also crystallized. 2013 . 1 and 2 represent graphically the data for histidine and tryptophane which are given/ in Table I.4 to 6.4. since this acidity was necessary for the condensation of methone with formaldehyde. Wadsworth and M. In slightly alkaline Downloaded from http://www. Jacobs for the opportunity to compare the two compounds.2.org/ by guest on May 30. the curves for reversibly bound formaldehyde are omitted. 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 3.5. in Alkaline Solutions-The experiments on the Equilibria reversal of the reaction by methone were all carried out in acid solution (pH 4. but under the conditions of our experiments the product of the reaction between histidine and formaldehyde did not separate from solution. Jacobs. This substance was synthesized by *Jacobs and Craig by the condensation of tryptophane and formaldehyde in acid solution (10).
0 112.6 150.8 37.6 98.0 47. 100 Cc :.7 138.0 125.4 7.jbc.0 23.0 116.O 35 .c Alanine 71.0 57.8 Glycine 72.0 28.: 147.0 118.2 97 . 2013 .3 14.2 8.5 48.0 165.0 58.0 8.0 8.0 34 .0 .5 66.2 25.0 55.0 N HCHO 39” N HCHO 39” HCHO 39” N HCHO 39” N HCHO 39” N HCHO 39” HCHO 39” N HCHO 39” 140.4 141 .7 70.0 8.0 139.8 Arginine 11.0 8.7 129.0 67.5 118.0 8.0 69.c 102.c’ 68.7 44.6 Guanidine 70.2 8.0 140.0 125.8 107.c 1 119.2 42.0 .: 112.0 140.8 -_ Cysteine* 37.7 65 .9 243 31.7 31. 0.2 27.8 40.0 Lysine Amino Free HCHO Amino Free HCHO Free HCHO Amino Free HCHO Amino Free HCHO Amino Free HCHO Free HCHO Amino Free HCHO 32.0 147.7 54.o 66.0 143.4 7.7 44.4 69.org/ by guest on May 30.0 105.0 147. 3 I 4 days 17 day) 11 da YS 28 days Results o.c I 105.TABLE 1 Analytical 2 hrs.O 117.3 82.2 71.0 Downloaded from http://www.O 95.c 6O.2 7. ldays i’days 11 day.0 148.c 1 137.0 110.0 108.4 136.0 165.2 60.8 Alanylglycine 70.2 24.2 52.0 22 .7 8.0 144.0 70.0 132.0 131.: 34.O 103.O 19.0 50.9 133.6 35.0 107.0 7.0 61. per.0 8.9 53 .7 60 .5 97.0 139.0 144.0 4.0 95.0 7.0 148.f Solution) 7- Inihl .0 56.0 65.0 144.5 148.0 142.4 - Glycylalanine 153.0 76.0 122.c 123.8 69. 10.f Reactivity of Amino Compounds with Formaldehyde (Mg.0 8. [nitial Final --~24 hrs.8 95.c 129.t 111.0 114.0 115.
c 15.5 85.8 47.6 105 .8 55.8 8.0 36.7 50.5 32. 3 These two experiments were carried out at 36-37” instead of 39”.3 30.8 22.5 8.2 45.5 48. 8 hrs.0 Tryptophane Amino N Free HCHO HCHO 39” Amino N Free HCHO HCHO 39” 42.2 71.4 37.1 6. 4 days -14 hrs.8 65.0 117.5 76.5 15.8 7. 10 min.0 ~-~~ 25.0 25.3 11.7 61.7 40.8 .4 28.5 95.1 18.5 66.6 58.6 62.7 Trace 8.8 50. 1 hr. The irregular results obtained later may be partly due to the instability of the acid.0 g s Downloaded from http://www.( 56. gives abnormal results in the Van Slyke analysis.5 61.5 6.4 22.4 29. t Glycylalanine.0 46.5 2.0 119.4 62.2 51.jbc.4 acid1 “ $ (2%) Amino ‘I I‘ N “ <L 61.2 93.2 45. is CD .2 7.0 8.0 61.0 46.org/ by guest on May 30. 5 hrs.2 27.4 Toxin Free HCHO HCHO 39” Amino N Free HCHO HCHO 39” 68.9 41.6 7.6 20.0 37.4 20.9 62.5 33.5 29.2 after 4 hours.0 57.5 77.o 118. In this experiment the pH at first fell rapidly and was readjusted to 8.5 54. * At this concentration of cysteine the reaction had reached equilibrium in less than 15 minutes.9 48. hence.4 33. 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.8 49.Aspartic Glutamic Peptone 7.7 84.6 2o.8 40.0 32. -0.2 94. -- 7.0 5.8 Histidine 15.0 38. like glycylglycine (9).0 -2 hrs.P 8.0 37. -13 hrs.0 53.0 60.0 24.0 7.7 75.9 39.( 8.5 47. the amino nitrogen figures are not reported.5 115.f 6.0 37.8 12.! 8.9 41.6 E Q 1.5 48.0 4 hrs.0 61.6 88.4 132.1 38.0 132.5 13.
055 0. 6.044 4 0.f 0 0 54 56 2: 2i 2( . 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 0.05 0.05 0.-mole per 1. In this experiment the pH at first fell rapidly and was readjusted to 8. 0.org/ by guest on May 30.025 0.03 0.048 0. gives abnormal results in the Van Slyke analysis.042 0.028 0.048 0.036 6.- b e e e E ss 00 c 4 -e 5 J : - A 4 $.029 0.2 after 4 hours.05 Amino HCHO Amino HCHO Amino HCHO ‘I M) N N N 6. 2013 .10 0.420 Formaldehyde Percentage Reaction TABLE with Amino Acids II Reacting - of Components - .037 71 0. The irregular results obtained later may be partly due to the instability of the acid.038 * At this concentration of cysteine the reaction had reached equilibrium in less than 15 minutes.I 3: 23 58 0 0 56 61 46 76 3( 7: ( <: 5f 6: 4: 71 li 1.043 0. 0. 1 Glycylalanine.040 0.05 0. like glycylglycine (9).05 0. J These two experiments were carried out at 3637” instead of 39”. GE 7: 8: 6( 31 7E 1 4 5E 6E 41 7: 3: 8: << .c 23 21 14 63 5: 61 5E 6: if 77 18 1E 15 If l( 2: 1: 2: 6. hence.049 7! 0.4 5.045 Glycine Alanine Alanylglycine Glycylalanine (0. Downloaded from http://www.05 0.05 0. Histidine Tryptophane Cysteine* Arginine Guanidine (0.044 0. 2. the amino nitrogen figures are not reported.jbc. l 83 2 -2:g g I?.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.
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.. tionships in alkaline solution can be predicted from the results of the methone reaction.. Alanine.A. t Average of two experiments. Lysine. 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. alanylglycine. in a mixture containing insufficient formaldehyde to react with all the amino groups.s -81 k 4 d 85’ 63 1 k c 4 4 rz ... Argininef.-- Cysteine Glycine.. 2013 . 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 - - - 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. Glycylalanine 4 2 ...org/ by guest on May 30. C. . Peptone.. for example.jbc. Toxin. will gradually remove formaldehyde from its compounds with such acids as cysteine and arginine. Wadsworth and M. This table represents the same series of experiments as Tables I and II. * Average of three experiments. This may serve as an example of the complex equilibria in a mixture of formaldehyde with crude toxin... Downloaded from http://www. and this will result in a decrease of reversibly bound formaldehyde in the mixture. . If the relaTABLE Percentage of Reversibly E c N III Bound - Formaldehyde . Guanidine Alanylglycine. this is recorded as a percentage of the total amount of formaldehyde which had reacted (initial value minus free formaldehyde value). it is evident that.
2 and allowed to react with equivalent quantities of formaldehyde for 24 hours at 39”.org/ by guest on May 30.432 Formaldehyde Reaction with Amino Acids 0. 0 1. One portion of each solution was 120 100 .4 z 20 b. The Downloaded from http://www. 1. The solutions were then readjusted to pH 8.1 M alanylglycine in phosphate the remainder was diluted to 0. Reaction formaldehyde mixed buffer.2. --Q---m- ---____ --_ 2 4 6 8 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. buffer at 39” with and and and an equal volume of 0.jbc.2 -. 2013 .8 01 i5 2 E . ’ : E 4 40 60 80 I .05 M with phosphate All solutions were incubated incubated as a control.6 $ I: I. analyzed at intervals as previously described.1 M solutions of cysteine and of arginine were prepared in phosphate buffer at pH 8.o I.
who Downloaded from http://www. It is interesting to compare this series of experiments with one reported by Holden and Freeman (l). Reaction of tryptophane formaldehyde then.org/ by guest on May 30. 2. We may safely assume. 8 E % 80 ! -1 60 . C. Pangborn 433 results are summarized in Table IV. Wadsworth and M. 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.jbc. g 40 : 20 2 4 6 8 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. 2013 . that the behavior of the different formaldehyde-amino compounds toward methone shows their relative stability in alkaline solutions.A. too .
05 M + cysteine 0. The acids which show the greatest affinity for formaldehydehistidine.2 8.‘j B Alanylglycine 0..org/ by guest on May 30. 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.6 92..5 * In this case the initial value is that found immediately after mixing the solutions of the two amino acids.: 83 92...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..05 M.2 “0 cd 12 :z$ 2 “b “b x -~-_ 3 . Arginine 0.6 8.6 I 86.) Equilibria in Alkaline jl a 3 .494 101 97 82 89 64. tryptophane.925. 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. tyrosine)-are those which have other functional groups in the molecule in addition to the amino and carboxyl groups.696 96 90 92. The second carboxyl group in aspartic Downloaded from http://www.. per 100 cc.. in the experiments of Holden and Freeman.. Obviously TABLE IV Solutions Reversibly bound HCHO (mg.. Cysteine 0. 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..813. Alanylglycine 0. it is possible that the same may be true at least in the initial stages of the other experiments.. 8.. after the cysteine and arginine solutions had reacted with formaldehyde for 24 hours.923..417..~-2 --~-- I I Time after mixing 05 OS + j $ $ N 07 d 86.05 M.05 M..2 8...556.941..jbc.2 8.05 M -k arginine 0.2 8.4 7. cysteine (and.4 73 65.560.376 176 38....05 M.2 7.620.. arginine. 2013 ..
Wadsworth and M. We suggest that. 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. and (c) a stable compound probably formed by further reaction or rearrangement of compound (b). With regard to the mechanism of the reaction. while by chemical methods we find only about 10 per cent combined even after 4 days at 39”. In studies of toxoid production it is evidently insufficient to draw a distinction between “free” and “bound” formaldehyde. under the experimental conditions described by us and. possibly a hydroxymethyl or methylene derivative. also during the production of toxoid. 2013 .A. Evidently the two sets of data do not represent the same reaction. In the case of arginine. there may be at least three stages in the combination of formaldehyde with amino groups: (a) a loosely associated molecular compound. are markedly less reactive than the two dipeptides prepared from them. C. Pangborn 435 and glutamic acids decreases the reactivity. It is interesting also to compare our results with those of Tomiyama (5). (b) a labile chemical compound.org/ by guest on May 30. The simple amino acids. although Sorensen (12) found that this group could not be determined by the form01 titration method. Since guanidine itself combined with formaldehyde under similar conditions. it may be recalled that Hahn and his coworkers (11) found evidence of two stages in the reaction between tryptamine and substituted pyruvic acids. A calculation from Tomiyama’s 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’. They considered the first step to be the formation of an addition compound which then loses water to form the carboline ring.jbc. therefore. the analytical data indicate 2 moles of formaldehyde reacting in 0. yielding the stable carboline acid. indicated by the reversible stage in our experiments. it seems probable that the guanidine group of arginine also reacts with formaldehyde. as the “bound” formaldehyde may be either reversibly or irreversibly combined. alanine and glycine. Downloaded from http://www.05 M solution for each mole of arginine. the molecular compound detected by physicochemical methods must be immediately dissociated when chemical methods of analysis are used.
. S. 759 (1936)... Exp. L. 4. Australian J.. Biol.. 9.. Australian J.. 289 (1932). 92 (1932).. 767 (1932-33). 2. G.. and Deakers. Ber. J. 12. 2013 . 3. J. 7. J. 340. M.. Biochem. W. Chem.. 8. W. and Werner. 123 (1905). Butler. M.436 Formaldehyde Reaction with Amino Acids while some of the “free” formaldehyde may be loosely associated with amino groups to form molecular compounds.jbc.. and Med. J.. Holden. Siirensen. Infect. 6. 2. A. BioZ. Ges. H. Chem.. Schales. 5. 217 (1932-33). reversible reaction.. E. Freeman. and Bystrenin. and Med.. Wadsworth. M. A. A. Biol. Z. T. F. 51 (1935). Fischer. Levy. 99... 7. SC. 11. BIBLIOGRAPHY 1. C.. Exp. irreversible one. A.. T. 620. Ann. L.. Dunn. BioZ. 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. Chem. The reversal of the reaction has been studied in both acid and alkaline solutions. Gubareff. Jacobs. W. O. 117 (1930).. 189 (1931). Dis. 266. S. Ann. and Wheeler. Chem. biol. Tomiyama. SUMMARY 1. 37. Biol. 10. J. M. Downloaded from http://www. L. M. 113. 107 (1935). 2486 (1994). Hahn. Sot. Biol. Z.org/ by guest on May 30. 99.. Biochem.. them. Velluz. E. and Craig. 111. Blirwald.. 66... H. rend. SC.. and Freeman. In most cases two stages in the reaction can be demonstrated: (a) a rapid. 123 (1934). L. 111.. (b) a slower. P. Chem. 45 (1908). Compt. Chem.. 7..... 8.
org/ by guest on May 30.jbc.Downloaded from http://www. 2013 .
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