the reaction of formaldehyde with amino

7/28/2019 The Reaction of Formaldehyde With Amino

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Augustus Wadsworth and Mary C. Pangborn

WITH AMINO ACIDS

THE REACTION OF FORMALDEHYDE

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

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THE REACTION OF FORMALDEHYDE WITH AMINOACIDS

BY AUGUSTUS WADSW ORTH AND MARY C. PANGBORN(From the Division of Labo ratories and Rese arch, New York State Departmentof Health, Albany)

(Received for publication, July 6, 1936)The studies here recorded form part of an investigation of thereactions that occur when diphtheria toxin is converted into toxoid

and may also prove of value in further studies of the action of toxinin the tissues. When formaldehyde is added to toxin, it reactswith the various amino compounds present in the mixture. Whilethe reaction between amino acids and formaldehyde has beenextensively studied, the experimental conditions employed haveusually been different from those which prevail in the formationof toxoid from toxin. The usual procedure at present for thepreparation of diphtheria toxoid is to treat the toxin with from0.3 to 0.4 per cent formalin (equivalent to 0.12 to 0.16 per cent offormaldehyde) at 37-39 for 4 weeks or more, the init ial reactionbeing in the range of pH 7.6 to 8.4. The optimum concentrationof formaldehyde varies with the medium in which the toxin isproduced and appears to be related to the amino nitrogen contentof the crude toxin; it is less than the amount theoretically equiva-lent to the amino nitrogen present. It is obvious that the resultsof experiments at high temperature, high pH, or high concentra-tions of formaldehyde have only an indirect bearing on the toxinproblem.

A few investigators have reported studies of the reaction under condi-tions more or less resemb ling those of toxoid formation. Holden andFreeman (1) incubated several amino acid s with 0.5 per cent formaldehydein 0.1 N sod ium hydroxide at 37 for 36 days. They found no reaction inacid solution, while in 0.1 N alkali there was a rapid initial reaction, fol-lowed by a further slow decrease in amino nitrogen. They also found thatthe simp le amino acid s were markedly less reactive than certain proteoseand peptone prep arations previously stud ied by Freeman (2). Gubareff

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424 Formaldehyde Reaction with Amino Acidsand Bystrenin (3) investiga ted the effect of varying initi al alkalinity andconcentration of formaldehyde on the speed and com pleteness of thereaction with glycine at 38. Levy (4) stud ied the titration curves of anumber of amino acid s at 30 and conclude d that the amino group maybe assoc iated with either 1 or 2 mo lecules of formaldehyde, but the com-plex containing 2 mole cules apparently forms only in the presence of alarge excess of formaldehyde. Tomiyama (5) determined equilibriumconsta nts for the reaction of formaldehyde with glycine, alanine, andproline at 25 in a pH range from 8.0 to 10.0 and found that a constantwas obtained only on the assum ption that the comb ining ratio was 1.Both Levy and Tomiyama consider that their data indicate the formationof molecular compou nds.

The purpose of the present study was to obtain data on thebehavior of amino compounds of known constitution when treatedwith formaldehyde under conditions similar to those used for theproduction of diphtheria toxoid. The following substances werestudied: (a) amino acids-glycine, alanine, lysine, cysteine, as-partic and glutamic acids, histidine, tryptophane, and arginine;(b) guanidine; (c) two synthetic dipeptides-alanylglycine andglycylalanine; and (d) peptone and a crude diphtheria toxin.

EXPERIMENTALThe amino acids and guanidine were purchased from the East-

man Kodak Company. The peptone used was Difco proteose.The toxin was a crude diphtheria toxin produced in infusion-freemedium prepared with the same peptone (6). Alanylglycine andglycylalanine were synthesized and purified according to themethods of Fischer (7).

MethodsThe substances o be studied were dissolved in 0.05 M phosphatebuffer and the hydrogen ion concentration was adjusted to the

desired level by the addition of 1 N sodium hydroxide. The pHrange employed was 7.8 to 8.4 (determined calorimetrically). Inmost cases the concentration of the test substance was 0.05 M.Formaldehyde was added in an amount approximately equivalentto the amino nitrogen present. The solution was then made up tovolume, mixed, and analyzed as soon as possible. When the re-action was very rapid, as in the case of cysteine, the initial valueswere determined by the analysis of control solutions. Toluene



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A. Wadsworth and M. C. Pangborn 425was added as a preservative, and the solutions were incubated inparaffin-sealed bottles at 39. At intervals (usually after 2 and 24hours, 7, 14, 21, and 28 days) samples were withdrawn and ana-lyzed 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 formalde-hyde were found to be stable on incubation, except in the case ofcysteine, which is gradually oxidized.

Free Formaldehyde-The method was essentially that recom-mended by Velluz (8) and depends on the fact that the precipita-tion 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 willbe described here. The quantities used were adapted to microand semimicro weighing.

The sample to be analyzed, containing from 0.4 to 2 mg. offormaldehyde, was mixed with 20 cc. of a saturated aqueoussolution of methone (4.5 gm. per liter). If necessary, 2 or 3 dropsof 10 per cent acetic acid were also added to bring the final pH ofthe mixture in the range 4.4 to 5.0, since it was found that in thepresence of amino compounds the pH must be 5.2 or lower to insurequantitative precipitation of free formaldehyde. After themixture had stood at room temperature for 4 hours, the precipi-tate was collected on a microfilter, washed with water, dried for 2hours at llO, and weighed. 1 mg. of formaldimethone is equiva-lent to 0.1027 mg. of formaldehyde.

Warm acetone was used to wash the precipitate from the filterafter weighing. When the solutions contained protein or peptone,it was found convenient to dissolve the wet precipitate from thefilter with warm acetone, remove the acetone in a water bath,resuspend the purified precipitate in water, then filter, dry, andweigh. In agreement with Velluz, we found the method to beaccurate to about 3 per cent.Reversibly Bound Formaldehyde--In order to study the reversalof the reaction between formaldehyde and amino compounds, theformaldehyde determination was modified. The solution to beanalyzed was mixed with methone and the mixture was kept at39 for 3 days before it was filtered and the precipitate weighed.Usually the amount of formaldimethone thus found was greater



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426 Formaldehyde Reaction with Amino Acidsthan that found in the free formaldehyde determination, indi-cating that exposure to methone at 39 had caused an appreciablesplitting of the formaldehyde-amino compounds which hadformed. The 3 day period does not necessarily give the maximumreversal possible with this reagent, since in a few cases longerperiods of standing at 39 gave slight further increases in theamount of formaldimethone precipitated. For comparative pur-poses, however, it was considered more important to adopt aconstant standardized procedure than to attempt a determinationof the maximum reversal

It was found in some control experiments with solutions ofglycine and peptone which had reacted with formaldehyde thatacidif ication of the reaction mixture to pH 4.8 with subsequentincubation at 39 was insufficient to reverse the reaction in theabsence of methone.

ResultsThe experimental data are given in Tables I and II. It will beseen that the compounds studied varied greatly in the speed andcompleteness of their reaction with formaldehyde. The differ-ences were, in general, in the same order as those found by Holdenand Freeman, but were somewhat more marked at the higherhydrogen ion concentration employed by us. On continued incu-bation of the amino compounds with formaldehyde there was asteady decrease in the percentage of the total combined formal-dehyde which could be split off by methone. There were fourexceptions: (a) the two dipeptides, with which no reversal of thereaction was detected at any test period; and (b) arginine andguanidine, with which there was no significant change in thepercentage of reversibly bound formaldehyde during the wholetime of incubation. Table III shows the variations in reversiblybound formaldehyde, calcuIated from the data of Table I. Fromthe gradual change in stability towards methone, it appears mostprobable that in these cases the reaction proceeds in two stages.The initial reaction may be quite rapid and is rather easily revers-ible. In the second stage, characterized by increased stabilitytoward methone, the init ial reaction product is probably trans-formed into a more stable compound by a secondary reaction orpossibly a rearrangement.



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A. Wadsworth and M. C. Pangborn 427These two &ages are further illustrated by the special cases of

histidine and tryptophane. Figs. 1 and 2 represent graphicallythe data for histidine and tryptophane which are given/ in TableI; 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 reactionapproaches completion. It can scarcely be supposed that the truecombining ratio varies in such a way, but it is quite reasonable toassume that the init ial reaction product is more stable towardmethone than toward nitrous acid (tryptophane) or vice versa(histidine), and that this init ial product is gradually transformedinto a second compound which is stable toward both reagents.In this case the apparent combining ratio would vary in themanner found by experiment.

The reaction product of tryptophane and formaldehyde crystal-lized from the solution and was identified as 3,4,5,6-tetrahydro-4-carbolined-carbonic acid. This substance was synthesized by*Jacobs and Craig by the condensation of tryptophane and formal-dehyde in acid solution (10). When our product was comparedwith a sample of the carboline acid obtained from Dr. Jacobs,the two substances melted simultaneously with decomposition at306 (uncorrected) and there was no depression of the meltingpoint when the two were mixed. The yield of the carbolinecompound under the conditions of our experiment was nearlyquantitative. The fact that this condensation takes place soreadily 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 trypta-mine at physiological temperatures in the pH range of 3.4 to 6.2.We are much indebted to Dr. Jacobs for the opportunity tocompare the two compounds.

Holden and Freeman state that formolized histidine also crystal-lized, but under the conditions of our experiments the product ofthe reaction between histidine and formaldehyde did not separatefrom solution.

Equilibria in Alkaline Solutions-The experiments on thereversal of the reaction by methone were al l carried out in acidsolution (pH 4.4 to 5.0), since this acidity was necessary for thecondensation of methone with formaldehyde. In slightly alkaline



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