THE PROTEIN-FORMALDEHYDE REACTION

I. COLLAGEN

BY EDWIN R. THEIS

(From the Biochemistry Division, Department of Chemistry, Lehigh University, Bethlehem)

(Received for publication, March 8, 1944)

A careful study of the literature relating to the reaction between the fibrous proteins and formaldehyde suggests that some divergence of opinion exists. Harris (11) and Birch and Harris (4) in 1930 showed that the titration of amino acids, with hydrochloric acid in the presence of formal- dehyde, is not affected, while the titration with sodium hydroxide is markedly affected. Harris explained this phenomenon as the repression of acidic groups upon acid titration and the repression of basic groups upon alkaline titration.

Using the zwitter ion concept of Bjerrum (5), we would then not expect formaldehyde addition to affect in any way the acid titration of a protein, while we would expect it to influence the alkaline titration, since in the alkaline zone the formaldehyde undoubtedly reacts in some manner with the available free and uncharged amino groups. Thus, this concept would lead us to expect little or no shift in the isoionic point of the protein upon treatment with formaldehyde.

Tomiyama (26) believes the anionic form of the amino acid reacts with the formaldehyde. He also considers the protein-formaldehyde reaction in terms of the electronic theory. He pictures the formaldehyde as a dipolar molecule, +CH&, and since the amino or imino group of the anionic form of the amino acid has 2 unshared electrons, the two compo- nents react to give the accompanying formula.

H H

C:;;: + +CHtO- - C:kC&O

ii ii

Levy and Silberman (15) have shown mathematically from their studies that 2 molecules of formaldehyde combine with 1 molecule of amino acid. Bergmann et al. (3) have isolated a triformyl compound and further shown that the triformyl derivative changes to the monoformyl upon addition of alkali.

Reiner and Marton (16) postulated the following reaction between pro- 87

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88 PROTEIN-FORMALDEHYDE REACTION. I

tein and formaldehyde,

NHa NHa.CHzO /

\

/

“\ + CH,O ---) R

coo \ COOH

the aldehyde being held to the amino group by secondary valence. Ein- hour (8) showed that the acid amides fix formaldehyde and suggested the following reaction, R-CONHS + CHZO + R-CONH . CHtOH. Cher- buliez and Fier (7), and later Bergmann, found that diketopiperazines react with formaldehyde, taking up 2 molecules of the aldehyde.

/“:: N-CH, OH o=c CH2

I I /\

+ 2CH20 + O=C CH2

H2C\NH/C=o

I I H2C c=o

\/ N-CH20H

Levy and Silberman (15) have taken exception to the interpretations of Tomiyama, maintaining that he made no distinction between amino and imino groups. Balson and Lawson (2) have suggested that the number of formaldehyde groups which can be introduced corresponds to the number of hydrogen atoms attached to the nitrogen atom and have therefore proposed Reactions 1, 2, and 3.

(1) =NH + CHtO -+ =N.CHtOH

(2) --NH2 + CH20 + -NH. CHeOH

CHzOH CHzO / / \

(3) -NH.CHaOH + CHzO --) -N + CHzO + -N \ \ TH2

CHzOH CHzO

Stiasny (17) has suggested that formaldehyde reacts with gelatin in pos- sibly two ways, in one, with the basic groups, changing them to neutral ones, and in the other, with the imino groups of the peptide linkage. He sug- gests that the first reaction proceeds through an intermediate formation of a triformyl derivative which t,hen changes to the monoformyl. In the second reaction, Stiasny postulates a binding of the formaldehyde with the weakly basic imino groups, forming methyl01 compounds,

R-CO-NH-R’ + CH(OH)t -+ R-CO-N-R’ + H20 I

CHzOH

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E. R. THEIS 89

Stiasny further suggests that the free amino groups of the gelatin react rapidly, while the peptide groups only do so gradually. He believes that the action of the formaldehyde on the basic groups is such that not only the acid and base fixation capacity is influenced but also that of the fixation of tanning materials and dyes.

Since 1936 a number of papers, dealing with the reaction of the fibrous proteins and formaldehyde, have appeared in the literature. Theis and Schaffer (24) studied the collagen-formaldehyde reaction through a quanti- tative measurement of the breakdown of the internal structural forces as indicated by the change in “shrinkage” temperature. This work was fol- lowed by several additional papers (23, 22, 20, 19) relative to the actual fixation of formaldehyde by collagen. In 1939 Highberger and Retzsch (12) and in 1940 Highberger and Salcedo (13) investigated this reaction in a comprehensive manner. Bowes and Pleass (6), Holland (14), and Gustavson (10) have also studied this reaction in recent years.

EXPERIMENTAL

Specially prepared collagen material was used for the experiments. The preparation of the collagen has been previously described (21). 2 gm. samples of the collagen were placed in bottles together with 200 ml. of 0.1 N KC1 solution made 1 per cent with respect to formaldehyde and then t,he series of samples was adjusted to definite hydrogen ion concentrations with either hydrochloric acid or sodium hydroxide. The range covered was from pH 1.0 to 13.0. The bottles and contents were placed in a thermo- stat maintained at 20” for 72 hours. At stated periods, the samples were agitated in order to promote equilibrium. After the 72 hour period the pH at equilibrium was determined by means of a Beckman glass electrode assembly; the protein material was removed and pressed several times between blotting paper at 10,000 pounds per sq. in. It has previously been shown that such pressure removes the free water and any free electro- lyte for all practical purposes. We, therefore, have assumed that all free formaldehyde is correspondingly removed, leaving behind only that which is firmly bound to the protein itself. After being pressed, the protein- formaldehyde compound was allowed to dry in air, and was then ground in a small Wiley mill to a 60 mesh powder. The material was then ready for analysis for nitrogen, for bound acid or base, and for fixed formalde- hyde. The met.hod used for the determination of formaldehyde is that of Highberger and Retzsch (12) described elsewhere. The methods used for the nitrogen and for bound acid or base have been previously de- scribed (21).

Fig. 1 shows the data obtained. The series of curves may be inter- preted as follows:

Curve A represents the regular acid or base fixation for the particular

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.6

PROTEIN-FORMALDEHYDE REACTION. I

% HCHO

0 .25

0 so 0 I:0 6 20 @ 30

0 50

8 ACID-BASE; BLANK

FIG. 1. Showing the acid, base, and formaldehyde bound by collagen treated with different amounts of formaldehyde over a wide pH range.

native collagen used, showing a maximum acid fixation of 0.87 milliequiva- lent per gm. of protein, a maximum base fixation of 0.38 milliequivalent, an isoionic point at pH 6.5, a plateau in the pH range 7.0 to 9.0 indicative

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E. R. THEIS 91

of the back titration of histidine and of such a-amino groups as may be present, and a sharp point of inflection at pH 10.0, beginning the back titration of the e-amino groups of lysine. This curve is identical in trend with those given in earlier work with the exception of the isoionic point which in this case is somewhat more acid.

Curve B represents the titration curve of the collagen-formaldehyde compound formed by the reaction of the collagen in a 1 per cent formalde- hyde solution. This curve is identical with Curve A in the pH range 0.8 to 9.0. There is no indication of a shift in isoionic point. In the pH range 9.0 to 11.0 more base is fixed than is the case for the native collagen, indica- tive of the reaction between the c-amino groups of lysine and formaldehyde. Curves A and B merge at pH 12.0 and approach a maximum value.

Curve C represents the acid or base bound by the collagen-formaldehyde compound formed in a 5 per cent formaldehyde solution. This curve is also identical with that of Curve A in the pH range 0.8 to 7.0 and definitely shows no shift in the isoionic point of the collagen. However, owing to the large excess of formaldehyde present during the reaction, more base is fixed in the pH range 7.0 to 9.0.

Curve D represents the formaldehyde fixed by collagen in a 1 per cent formaldehyde solution over a wide pH range. These data indicate about 0.15 milliequivalent of formaldehyde fixed at pH 1.0, thereafter increasing almost as a straight line function to 0.43 milliequivalent at pH 6.5 or at the isoionic point. At the isoionic point, there is a very definite break in the curve, the fixed formaldehyde increasing to approximately 0.5 milli- equivalent and then remaining essentially constant from pH’7.0 to 9.5. At pH 9.5 there appears another break, the fixed formaldehyde increasing sharply at this point and continuing up to pH 11.5, at which point approxi- mately 0.87 milliequivalent of aldehyde is bound. An apparent break in the curve occurs at pH 11.5, indicative of another reaction.

Curve E represents the formaldehyde fixed by collagen from a0.25per cent aldehyde solution. The curve shows a marked break at pH 9.5 and only a slight indication of a plateau region, but gives approximately the same aldehyde fixation at pH 12.5 as does Curve D.

Curve F represents the formaldehyde fixed by collagen from the 0.5 per cent solution. This curve shows a slightly lower aldehyde fixation in the acid zone compared with Curve D, a plateau in the pH zone 8.0 to 9.5, and approximately the same aldehyde fixation as is shown by Curve D at pH values greater than 9.5.

Curves G, H, and I represent formaldehyde fixation by collagen at the higher concentrations of formaldehyde; i.e., the 2,3, and 5 per cent formal- dehyde solutions. Curves G and H show a definite point of inflection and even indications of a plateau in the pH range 6.5 to 8.5. These curves

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92 PROTEIN-FORMALDEHYDE REACTION. I

show a decided increase in aldehyde fixation at pH 12.0. Curve I shows a much greater aldehyde fixation in the acid zone, with a definite point of inflection at pH 7.0 and a maximum value of 1.6 milliequivalents of formal- dehyde fixed at pH 12.0. Curves G, H, and I all show a definite decrease in aldehyde fixation at pH values greater than 12.0, possibly due to decrease in formaldehyde concentration because of the Cannizzaro reaction of form- aldehyde itself at strong alkaline reactions.

DISCUSSION

Highberger and Retzsch, in explanation of their data, claim it is signifi- cant that the break, at pH 7.0 to 8.0 in their pH formaldehyde .fixation curves, occurs at a formaldehyde fixation slightly over 0.4 mM per gm. of collagen. This value, they claim, is close to the amount of lysine believed to be present in collagen. These investigators state that this particular break represents the equivalence point in the reaction of 1 molecule of formaldehyde with each free amino group provided by the lysine residues and, therefore, a priori this is indicative that only the undissociated amino groups are involved in the reaction. They further postulate that the increase in formaldehyde fixation at pH values greater than 8.0 represents fixation with the stronger basic guanidino groups of arginine. This argu- ment is advanced in spite of the fact that the pKs values of arginine and lysine are 12.5 and 10.5 respectively. They also point out that greater concentrations of formaldehyde cause a reaction between the excess formal- dehyde and the imino linkages owing to their lesser basicity.

The experimental data given in this paper are not in line with those obtained by either Highberger and Retzsch or by Bowes and Pleass. High- berger and Retzsch show a possible but indefinite plateau zone in the pH range 7.0 to 8.0, while the writer shows a clearly defined plateau in the pH zone 6.9 to 9.4. The explanation given by Highberger and Retzsch is that at the particular break in the curve the e-amino group of lysine has completely reacted with formaldehyde. The data given herein do not in any way support such an interpretation. The data given by Bowes and Pleass show a well defined maximum at pH 1.5, a minimum at pH 3.5, a constant fixation or plateau region at pH 5.0 to 11.0, a slight depression in fixation at pH 11.0, which they claim is real, and a sharp increase in fixa- tion at pH 12.0. The data obtained by Highberger and Retzsch and by Bowes and Pleass are quite different from those given by us. The differ- ence is due undoubtedly to the experimental methods used. After treat,ing the collagen with formaldehyde, Highberger and Retzsch thoroughly washed the collagen-formaldehyde compound either with water or with dilute sodium bisulfite solution. The writer believes that this washing gave rise to erroneous and erratic results and that the final picture as obtained by Highberger and Retzsch does not represent the true one.

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E. R. THEIS 93

It must be borne in mind that the protein-formaldehyde reaction is a reversible one, the protein-formaldehyde compound being readily affected by any changes in hydrogen ion concentration, formaldehyde concentration, or other external conditions. That this is true can be readily seen from Table I, which shows the change in fixed formaldehyde of the collagen- formaldehyde compound formed in a 1 per cent formaldehyde solution at pH 11.0, (a) when it is placed in water at pH 8.0, (b) when it is placed in water at pH 2.0, and (c) when it is heated at 105” for 12 hours. These data show definitely that the collagen-formaldehyde compound is to a large extent reversible. Therefore, washing the compound after treatment with water can only yield erroneous values for fixed formaldehyde.

As an interpretation of the data given in Fig. 1, the writer suggests that in the pH range 1.0 to 6.4 the formaldehyde reacts with the slightly basic

TABLE I Formaldehyde Fixation Reversibility

Treated at pH ll.Ot.. . . . . . . . 0.85 -0.33 ,I L‘ IL 11.0, then placed in water at pH 8.0.. 0.47 -0.11 ‘I Lt I‘ 11.0, “ “ I‘ ‘I “ I* 2.0.. . 0.29 +0.94 “ ‘I Ii 11.0, “ heated at 105”. . . . . . . . . . . . 0.70 -0.34

CHzO’ H+ or OH-:

* 1 per cent CHzO at pH 11.0 for 72 hours. t Millimoles of CH20, H+, or OH- fixed per gm. of collagen.

imino groups present in the peptide chains in such a manner as to form linkages or bonds between the polypeptide chains.

’ ‘+CHO ’ /

NHHN / \ 2 -+ /N-cHp-N\

+ He0

In this manner additional cohesive bonds or bridges are built up, thus giving increased resistance to contraction or shrinkage. At pH 6.4, formal- dehyde reacts with histidine, giving rise to the plateau zone at pH 6.9 to 9.4. The writer is well aware that there exists some difference of opinion whether the imidazole group of histidine reacts with formaldehyde (9). However, the present investigation appears to lend support to the view that such a reaction may take place, since in this particular pH range histidine is normally titrated back, as can be seen from a study of the acid-base binding data for collagen. As the pKs value of lysine is ap- proached, we might expect the e-amino group of lysine as it changes from -NH3+ to -NH2 to react with formaldehyde. This appears to be the fact, as a study of Curve D, Fig. 1, shows that at pH 9.2 increasing formal- dehyde fixation occurs. This increase is positive and approaches a defi-

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94 PROTEIN-FORMALDEHYDE REACTION. I

nite point of inflection at pH 11.5. The increase in formaldehyde fixation between pH 9.2 and 11.5 is about 0.40 mM per gm. of collagen and approxi- mates the lysine content (0.38 mM per gm.) of collagen. Such data and their interprebation indicate that the very basic guanidino group of arginine does not react to any great extent in the pH range studied. The writer believes, however, that if it were possible to study the reaction of collagen with formaldehyde at pH values greater than 12.0 we would find a further increase in formaldehyde fixation; i.e., a binding with the guanidino group of arginine. The Cannizzaro reaction prevents such an investigation.

Curve I (Fig. 1) represents data secured when a large excess of formalde- hyde is present during the reaction. These figures show that in the pH range 1.0 to 7.0 formaldehyde fixation is increased and must be due t,o a mass action effect, the aldehyde in all probability combining with a greater number of the weakly basic imino groups of the polypeptide chain. In the p%I range 7.0 to 12.0, about 0.80 mM of formaldehyde has become fixed. This value represents approximately twice the lysine content of collagen and thus leads us to believe that 2 molecules of formaldehyde are fixed by each undissociated amino group of lysine,

CHzOH

/ -NH* + 2CHzO ---) -N

\ CH,OH

forming a dimethylol compound. Curves G and H, representing data for the collagen-formaldehyde compound formed upon treatment of collagen with 2 and 3 per cent formaldehyde solution, show in general the same trend. These two curves merge with Curves D and F at pH values less than 6.0, show a plateau at pH 6.5 to 9.0, and a sharp increase in formalde- hyde fixation up to pH 12.0. Curve E represents data for the collagen treated with 0.25 per cent formaldehyde solution and shows only a slight indication of a plateau zone, but there is a definite indication of reaction of formaldehyde with histidine. Curves D, E, and F show approximately the same trend and amount of formaldehyde fixed in the pH range 9.5 to 12.0, indicative of a stoichiometric chemical reaction; i.e., the fixation of 1 mole of formaldehyde with each e-amino group of lysine.

In previously published work, Theis et al. (25) have postulated that, in the pH range 1.0 to 6.0, formaldehyde binds with the weakly basic imino groups of the polypeptide chains and not with the E-amino group of lysine. It is certainly to be expected that with this preparation of collagen, having an isoionic point of 6.4, the basic groups would exist for the most part in the charged state at any pH value less than 5.0. Under such conditions, the basic groups of lysine and arginine would exist in the charged ionic

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E. R. THEIS 95

form and the electronic pair of the nitrogen atom would not be available for formaldehyde fixation since it already is coordinated with the hydrogen atom. Thus the accompanying reaction should not t.ake place, since in that

H H H I-1

R:i:H + ;=‘:O . . . .

- R:N:C:O + H+

ii ii . . . . HH

.case the ability to bind H+ ions would be affected. A study of Curve A of Fig. 1 shows that the H+ ion-binding capacity of collagen is unaffected by form- aldehyde fixation. The preceding interpretation is supported by Atkin (1). In a discussion of his investigation dealing with the deamination of col- lagen, he states, “As a consequence we should expect the part of the curve corresponding to the back titration of the basic groups of lysine to dis- appear. This is between pH 9.0 and 10.0 and it is evident that in de- aminized collagen this part of the curve has disappeared.” In Curves D, E, and F of Fig. 1, the part of the curves representing the back titration of the lysine has not disappeared.

Theis and Esterly (20).have in the past used “shrink temperature” as a criterion for protein stabilization. “Shrink temperature” has been defined as the point at which the increasing disruptive tendencies exceed the dimin- ishing cohesive forces; thus the “shrink temperature” is actually a measure of the structural strength of the collagen expressed in arbitrary units. Since x-ray data for collagen have shown that this protein exists in the native state as an extended polypeptide chain, it is evident that the chain may contract upon itself. Collagen in the moist state shows a shrinkage temperature of approximately 58”. If the collagen is treated with reagents which enter into combination with its reactive groups, this shrinkage tem- perature may decrease or increase. Collagen treated with formaldehyde at various pH values shows an increase in shrinkage temperature at prac- tically all pH values. Fig. 2 shows such data for this particular collagen. Curve A represents the shrinkage temperature of native collagen, merely treated with aqueous acid or alkali. This curve indicates a shrinkage tem- perature of 57-58” in the pH range 6.0 to 9.5. At pH values less than 6.0 or greater than 9.5 decreased structural stability is evident. Curve B represents data for collagen treated at various hydrogen ion concentrations but in this case the acid or base solutions contained 0.5 per cent of form- aldehyde. A striking difference is apparent. At all pH values the shrink- age temperature has definitely increased. This increase is particuhlrly notable at pH values greater than 4.0. There are definite points of inflec- tion at pH 7.0 and pH 8.0. Curve C represents data for a 1 per cent formaldehyde solution, while Curve D is that for a 5 per cent formaldehyde solution. Curve C shows a slight increase in shrinkage temperature over

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96 PROTEIN-FORMALDEHYDE REACTION. I

that shown by Curve B and in line with the curves given in Fig. 1. In the pH range 1.0 to 6.0, Curve D shows a decided increase in structural sta- bility over that shown in either Curve B or C, again in line with formalde- hyde fixation data given in Fig. 1. It is to be particularly noted, however, that in the zone pH 8.0 to 12.0 all three curves merge. Such trends would seem to indicate two different and distinct chemical reactions, the one taking place over practically the whole pH range being especially noticeable

90.

80.

Formaldehyde treated Collagen

30

0 2

\ I

4 6 2 10 12 #I value of Treatment

FIG. 2. Comparison of the shrinkage temperature df native collagen with that of formaldehyde-treated collagen.

on the acid side of the isoionic point, and the other taking place at pH values greater than 8.0. This series of curves for shrinkage temperature lends support to the suggestion that, in both the acid and alkaline zones, it is the reaction of formaldehyde with the weakly basic imino groups of the polypeptide chains that gives to the collagen its thermolability as measured by the shrinkage temperature. In the alkaline zone, the for- maldehyde undoubtedly binds with the free basic groups of lysine in addi- tion to the imino groups.

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E. R. THEIS 97

SUMMARY

The collagen-formaldehyde reaction has been discussed in detail. It has been shown that the fixation of formaldehyde with collagen in no way affects the acid-binding capacity of collagen but does affect the base- binding capacity. No shift in the isoionic point could be demonstrated as due to formaldehyde fixation. Correlation between data for shrinkage temperature and formaldehyde fixation is shown.

BIBLIOGRAPHY

1. Atkin, W. R., Stiasny Festschrift, Darmstadt, 13 (1937). 2. Balson, E. W., and Lawson, A., Biochem J., 30,1257 (1936). 3. Bergmann, M., Jacobsohn, M., and Schotte, H., 2. ph2/sioZ. Chem., 131,lS (1923). 4. Birch, T. W., and Harris, L. J., Biochem. J., 24,108O (1930). 5. Bjerrum, N., 2. physiol. Chem., 104,147 (1923). 6. Bowes, J. H., and Pleass, W. B., J. Internat. Sot. Leather Trades Chem., 23,365,

451, 499 (1939). 7. Cherbuliez, E., and Fier, E., Helv. chim. acta, 6, 678 (1922). 8. Einhour, A., Ber. them. Ges., 41, 24 (1908). 9. Gerngross, O., and Gorges, R., Collegium, 391,398 (1926).

10. Gustavson, K. A., J. Znternat. Sot. Leather Trades Chem., 24,377 (1940). 11. Harris, L. J., Proc. Roy. Sot. London, Series B, 96,442,500 (1923). 12. Highberger, J. H., and Retzsch, C. E., J. Am. Leather Chem. Assn., 33,341 (1938);

34, 131 (1939). 13. Highberger, J. H., and Salcedo, J. S., J. Am. Leather Chem. Assn., 36, 11 (1940);

36,271 (1941); 37,276 (1942). 14. Holland, H. C., J. Znternat. Sot. Leather Trades Chem., 23, 215 (1939). 15. Levy, M., and Silberman, D. E., J. Biol. Chem., 118,723 (1937). 16. Reiner, L., and Marton, A., Kolloid-Z., 32, 273 (1923). 17. Stiasny, E., Gerbereichemie, Dresden (1931). 18. Theis, E. R., J. Am. Leather Chem. Assn., 37, 499 (1942). 19. Theis, E. R., and Blum, W. A., J. Am. Leather Chem. Assn., 37,553 (1942). 20. Theis, E. R., and Estcrly, A. R., J. Am. Leather Chem. Assn., 36,563 (1940). 21. Theis, E. R., and Jacoby, T. F., J. Biol. Chew!., 146, 163 (1942); 148, 105, 603

(1943). 22. Theis, E. R., and Ottens, E. F., J. Am. Leath rr Chem. Assn., 36, 330 (1940); 36,

22 (1941). 23. Theis, E. R., and Priestly, W., J. Am. Leather Chem. Assn., 34, 566 (1939). 24. Theis, E. R., and Schaffer, E. J., J. Am. Leather Chem. Assn., 31,515 (1936). 25. Theis, E. R., and Steinhardt, R. G., Jr., J. Am. Leather Chem. Assn., 37, 433

(1942). 26. Tomiyama, T., J. Biol. Chem., 111, 51 (1935).

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Edwin R. TheisREACTION: I. COLLAGEN

THE PROTEIN-FORMALDEHYDE

1944, 154:87-97.J. Biol. Chem. 

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