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Volume 79, 1951
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The Reaction of Hydrogen Chloride with Dry Proteins
Part 2—Collagen, Silk Fibroin, Elastin

[Read before the Otago Branch, November 14, 1950; received by the Editor, November 30, 1950]

In Part 1 of this series (Green, 1950) it was shown that the quantity of hydrogen chloride held in firm combination by dry collagen or silk could not be accounted for by the proportions of arginine, histidine, lysine, and hydroxylysine side chains in the proteins. To explain the presence of the excess acid, it was shown to be sufficient to postulate reaction with the aliphatic hydroxyl groups of the serine, threonine and hydroxylysine residues. This paper describes further tests of the hypothesis, by a detailed examination of the reactions of silk fibroin and elastin with hydrogen chloride, and by acetylation of some of the products of the reactions.

Experimental

Materials

Pure silk fibroin was kindly supplied by Dr. S. G. Smith, of the Shirley Institute, Manchester.

The elastin was the same as that used in studying water vapour adsorption (Green, 1948) and was ground to pass a 40-mesh but not a 60-mesh sieve. In view of the repeated extractions with boiling water involved in its preparation, it is unlikely that this specimen of elastin contained any measurable amount of collagen or gelatin.

The reaction of hydrogen chloride with silk fibroin and elastin

The reaction between silk and HCl was more rapid than that previously observed with collagen, and it became necessary to make measurements for fairly short reaction times. This rendered a long desorption period undesirable, and so the methods of Part 1 were modified by omitting the room temperature desorption stage for the product of the gaseous HCl reaction, and, in the ethereal HCl experiments, by washing the product with four changes of dry ether in quick succession before heating in vacuo. In order to reduce the speeds of reaction, the saturated (approximately 6·5 M) ethereal HCl of Part 1 was replaced here by a 0·2 M solution.

The behaviour of both silk and elastin was generally similar to that of collagen. In gaseous hydrogen chloride, each took up large quantities of the gas, the greater part being loosely held and easily desorbed. Each protein retained some HCl even on heating at 100° in vacuo over calcium oxide; and this combined HCl increased with time of reaction to a saturation value of 2·9% for silk and 2·8% for elastin. With silk in dilute ethereal HCl, the greatest quantity of combined HCl measured was about 2·5%, but, with more concentrated

[Footnote] Present address: University of Malaya, Singapore.

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solutions, samples containing over 2·6% were prepared, and it seems certain that this series was tending to the same saturation value of 2·9%. The results are given in detail in Tables I–III.

Table I—Reaction of Silk Fibroin with Gaseous Hydrogen Chloride
Time in HCl (hours) Total gain in weight Final gain in weight (g. per 100 g. silk fibroin) Combined HCl
¼ 1.24 0.18 0.47
½ 3.80 0.25 0.82
1 4.51 0.61 0.88
2 5.88 0.28 1.22
3 7.87 0.52 1.39
4 9.35 0.91 1.56
5 9.07 0.51 1.63
6 10.00 1.05 1.75
8 12.02 1.23 1.89
16 13.80 1.04 2.11
38 15.50 0.81 2.32
195 16.60 1.30 2.61
2164 17.30 2.90
Table II—Reaction of Silk Fibroin, with 0.2 M Ethereal, Hydrogen Chloride
Time in HCl (hours) Combined HCl (g./100 g. silk) Time in HCl (hours) Combined HCl (g./100 g. silk)
1 0.19 48 0.75
2 0.25 72 0.82
4 0.28 118 1.13
6 0.27 144 1.32
10 0.29 166 1.51
15 0.35 235 1.62
18 0.40 308 1.91
24 0.50 610 2.47
35 0.63
Table III—Reaction of Elastin with Gaseous Hydrogen Chloride
Time in HCl (hours) Total gain in weigt Final gain in weight (g. per 100 g. elastin) Combined HCl
2 17.8 0.92 1.32
6 26.2 1.48 1.89
15 28.8 1.58 2.03
30 29.0 1.70 2.12
47 29.1 1.70 2.32
70 29.0 1.95 2.44
91 28.9 2.21 2.51
121 29.1 2.28 2.58
140 29.5 2.20 2.66
218 29.1 2.31 2.76
382 29.2 2.35 2.80
1726 30.3 2.48 2.80

Acetylation

As the dry proteins reacted extremely slowly with cold acetic anhydride, either in the presence or absence of pyridine, silk and collagen and their hydrochlorides, prepared by the action of ethereal HCl, were dried at 100° in vacuo and acetylated by boiling 1 g. samples for thirty minutes with acetic anhydride. The suspension was then cooled and diluted with several volumes of dry ether to precipitate any of the protein which had dissolved. The product was filtered,

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washed several times with dry ether, allowed to stand over-night in the same solvent, filtered and washed with more ether, and finally dried at 100° in vaecuo and weighed.

Analysis of the acetyl derivatives was based on the fact that O-acetyl groups are readily hydrolysed by cold dilute alkali, while the N-acetyl groups are unaffected (Hendrix and Paquin, 1938). The methods used were similar to those of Blackburn and Phillips (1944), but modified to allow for the presence of titratable HCl in some of the samples.

The titrable acid was determined by covering a 0·5 g. sample with 50 ml. 0·05 N NaOH, protected from the air, for 24 hours. The mixture was then titrated to pH 7 with 0·05N H2SO4, using the glass electrode. In this titration, slowness of diffusion between the solutions within and without particles of insoluble protein introduced some uncertainty, which was eliminated by passing over the end-point several times in each direction at intervals of about thirty minutes. As N-acetyl groups are stable to the above treatment, the mixture from that titration was next analysed for total acetyl. 20 ml. 5N H2SO4 was added and the mixture distilled until its volume had been reduced to about 20 ml. It was then boiled under reflux for two hours and steam distilled with protection from the carbon dioxide of the air. When another 200 ml. had been collected, the two distillates were combined and titrated with 0·05N NaOH.

The HCl content of the same 0·5 g. sample was determined by gravimetric analysis of the residue in the distilling flask. Since it had been found that all the combined HCl held by the proteins could be titrated with dilute alkali, the O-acetyl was represented by the difference between the first titration and the HCl content; while the N-acetyl was the difference between total and O-acetyl.

As was to be expected in view of the high temperature (137°), it was found that some protein was decomposed during acetylation, so that theoretical yields were never realised. The net recovery for the proteins themselves was about 105%, corresponding to 95–97% of the theoretical yield; but the loss from the hydrochlorides was greater, so that the yield ranged from 95% for some silk hydrochlorides to as low as 88% for one collagen hydrochloride. At the same time, a disproportionately large loss of hydrogen chloride occurred, often leaving the acetyl derivative with only two-thirds of the original HCl content. Calculation of the results was consequently rather involved. In the O-acetyl determination, a correction was first applied for the base-binding capacity of the protein contained in the derivative. This was evaluated by carrying out a blank titration on the substance before acetylation and subtracting the equivalent of any HCl it contained. This blank was found to vary slightly from one hydrochloride to another. The O-acetyl titre was next corrected by subtracting the equivalent of the final HCl content of the derivative. Lastly, it was assumed that the protein lost during acetylation had the same composition as that remaining undecomposed, and the O-acetyl equivalent was calculated for 1 g. protein in the original sample.

The total acetyl figure was found to differ surprisingly little from the O-acetyl, implying that little or no N-acetylation had taken place.

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We therefore reserve this question for more detailed study and in Table IV report the O-acetyl data only. In spite of the rather complex nature of these computations, we believe them to be reliable, since extending the period of acetylation from one-half to one hour increased the O-acetyl content slightly, but did not sensibly affect the difference between the O-acetyl figures for silk and a hydrochloride.

Table IV—Acetylation of Silk Fibroin, Collagen, and their Hydrochlorides
(Quantities are given in milli-equivalents per gram original protein)
Starting material Initial HCl content Carbon-bound Cl (theory) Time of acetylation (hours) O-acetyl Difference form O-acetyl for parent protein
Silk fibroin 0 0 ½ 1.65
1 1.79
Silk hydrochloride 0.30 0.23 ½ 1.45 0.20
0.63 0.56 ½ 1.08 0.57
0.63 0.56 1 1.28 0.51
0.69 0.62 ½ 0.96 0.69
0.79 0.72 ½ 0.96 0.69
Collagen 0 0 ½ 2.11
Collagen hydrochloride 0.45 0 ½ 2.13 −0.02
1.28 0.38 ½ 1.80 0.31
1.37 0.47 ½ 1.67 0.44
1.50 0.60 ½ 1.56 0.55

Discussion

It is clear that the ability to form stable hydrochlorides, capable of retaining their combined hydrogen chloride even at 100°, is not confined to collagen, but is a general property of proteins. The single observation, reported in Part 1, for the maximum uptake of combined hydrogen chloride by silk fabric has been confirmed here by the value of 2.90% for pure silk fibroin.

On the other hand. Czarnetzky and Schmidt (1934) found a break in the gaseous titrntion curve, which they attributed to a compound of silk fibroin with 40 × 1015 moles HCl per gram (1·46%), having a dissociation pressure of approximately 14 mm. at room temperature. Since we have reported in Tables I and II compounds, prepared by two different methods, containing more than this amount of HCl and with a negligible dissociation pressure even at 100°, it is relevant to inquire into the reason for this conflict of evidence.

A major difference between the two experimental methods lies in the fact that Czarnetzky and Schmidt gradually increased the pressure of HCl up to one atmosphere, but, except in the case of gelatin, made no claim to have traversed the titration curve in the opposite direction by withdrawing HCl; whereas, in order to isolate our derivatives, we were obliged to carry out complete desorption at an elevated temperature. That this high temperature was not responsible for the firm fixation of HCl, however, is shown by the dependence, for all the proteins examined, of the quantity of combined HCl on the time of exposure to the gas or ethereal solution in the cold. If the formation of our stable compounds took place during the subsequent period of heating, their composition should be unaffected by the duration of the room-temperature reaction.

We are forced to the conclusion that proteins in general, and silk fibroin in particular, do take up cold gaseous HCl to form compounds

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with a negligible dissociation pressure. The reaction is a slow one and may be influenced by the quantity of adsorbed HCl present. At an HCl pressure of 14 mm. the amount of gas adsorbed would be small, and the rate of formation of a stable compound might well have been indiscernible in the experiments of Czarnetzky and Schmidt. The break they observed in the titration curve doubtless indicated the presence of a more labile compound with weakly basic groups, but did not represent true equilibrium, since we have shown that increasing the pressure to atmospheric and reducing it again to zero leaves the protein with some firmly bound HCl.

We have suggested in the previous paper that the figure of 2·9 g. HCl per 100 g. silk could be explained by assuming that hydrogen chloride reacted with each basic side chain and with the β-hydroxyl groups of approximately one-half of the serine and threonine residues known to be present. The probability that half of the serine and threonine residues were masked in the fibroin molecule had already been established by the work of Gordon, Martin and Synge (1943). It was further suggested that the reaction with the basic groups should be much more rapid than that with hydroxyl; and it was shown for collagen that the latter reaction rate approximately obeyed a uni-

Picture icon

Fig, 1—Plots of log(a—x) against time.

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molecular equation, being proportional at any time to the number of—OH groups still unreacted. In this way the quantities of hydrogen chloride taking part in the two reactions could be distinguished. The more detailed treatment of the silk reactions considered here provides a good test of the hypothesis, since silk fibroin differs from collagen in containing very few basic side chains. This test is illustrated in Fig. 1 by plots of the data of Tables II–III for silk and elastin in the forms of log (a—x) against time, where a represents the saturation value of combined acid, and x is the combined acid at time t.

The ethereal HCl reaction yields a good linear plot which can be extrapolated at approximately zero time to a point corresponding to × = 0·30. This can be interpreted as denoting the presence of basic groups capable of rapidly binding 0·30 g. HCl per 100 g. silk, and hydroxyl groups equivalent to the remaining 2·6 g., in good agreement with the respective values of 0·24 and 2·56 deduced in Part 1 from the composition of the protein.

The reaction of gaseous HCl with silk has not been included in the figure, as it is evidently of a higher order and the logarithmic plot is far from linear. No transformation of the co-ordinates was found which could fit the data to the common equations of reaction kinetics. This is less surprising than the fact that straightforward mathematical treatment has succeeded in deriving a simple formula for any of the reactions between HCl and such complex substances as fibrous proteins. Nevertheless, the data of Table I were fitted to an empirical straight line by plotting combined HCl against total HCl adsorbed. No explanation is advanced for this relationship, although it is conceivable that the aliphatic —OH groups react with adsorbed and not with gaseous HCl; but it is interesting to note that extrapolation to zero adsorbed HCl, that is, to zero time, yielded a value of about 0·2 g. combined HCl per 100 g. silk. We may say, then, that the reactions summarised in Tables I and II show that the initial rapid combination with HCl is of the order of 0·2–0·3%, in close agreement with theory.

The discrepancies shown in Table I between combined HCl and the gain in weight of the protein are rather erratic. They probably owe much of their irregularity to the difficulty of defining a dry protein or a dry hydrochloride, but they are at least of the right order of magnitude to be explained as loss of water from the hydroxyl reaction. Indeed, a plot of the observed loss (y) against combined hydrogen chloride (x) gives a fairly good straight line defined by the regression equation

y = 0·52x + 0·03

with a correlation coefficient of 0·80, The theoretical relation gives the equation

y = 0·49x − 0·12

It is seen from Fig. 1 that the data for elastin in gaseous HCl also give a linear plot of log (a—x) against time, extrapolating at zero time to a value of 0·94 for x. In terms of the theory which we have applied to collagen and silk, this implies aliphatic hydroxyl groups equivalent to 0·94 g. HCl and basic groups equivalent to 2·80 – 0·94 = 1·86 g. HCl per 100 g. elastin. This protein has been less completely analysed than many others, but Bowes and Kenten (1949) have recently pub-

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lished a table of amino acids in elastin which accounts for approximately 90% of the total nitrogen. No serine is reported in this table, but the presence of 2·7% threonine, with an HCl equivalent of 0·83%, substantiates our estimate of 0·94% for the HCl equivalent of the aliphatic hydroxyl groups. No histidine and lysine and very little arginine have been found in elastin, and we venture to suggest that unless it contains an unusually high proportion of terminal amino groups, a more complete analysis of this protein may lead to higher values for the basic amino acids more in conformity with its capacity for binding gaseous hydrogen chloride.

Acetylation

Boiling for thirty minutes with acetic anhydride introduces into silk rather less and into collagen rather more than the expected number of O-acetyl groups, based on the proportions of serine, threonine, hydroxylysine, hydroxyproline, and tyrosine in the proteins. The degree of O-acetylation of silk increases with time, and it is likely that shorter periods would yield a value consistent with the supposition that half the serine and threonine residues are masked; while longer periods might result in complete acetylation of all the hydroxyl groups. The hydroxyl radicals of serine and threonine are in the β-position and are thus closer to the polypeptide chain than any other polar side-chain groups. This may account for some of their unusual properties.

Comparison of the O-acetyl figures for silk and collagen hydrochlorides with those for the parent proteins reveals that they are best explained by the hypothesis advanced in Part 1 of this series. The basic groups of silk and collagen are respectively equivalent to 0·07 and 0·90 milli-equivalents of acid per gram protein. Any chlorine in excess of this has been introduced into the molecule by the reaction of aliphatic hydroxyl, e.g.

Therefore, every equivalent of chlorine in excess of the base-bound chlorine can be expected to reduce the O-acetyl content of the protein by one equivalent: The remarkable agreement between the second and fifth columns of Table IV demonstrates that this relationship does hold. Serine and threonine are the only hydroxyamino acids common to silk and collagen, so it is evident that the reduced O-acetylation of the protein hydrochlorides is bound up with the replacement of hydroxyl groups in these residues. Hydroxylysine probably behaves similarly. The only other possible interpretation of the table, namely, that the presence of hydrogen chloride in some way reduces the rate of reaction with acetic anhydride, is refuted by the result obtained when the period of acetylation of a silk hydrochloride was extended to 1 hour.

The loss of combined HCl during acetylation does not invalidate our conclusions, since there is no water present, and carbon-bound chlorine is presumably eliminated as HCl, with the introduction into the side chain of double bond incapable of being acetylated.

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Elementary analysis

Reaction between aliphatic hydroxyl radicals and HCl causes the elimination of an equivalent quantity of oxygen in the form of water, so that an examination of the elementary composition of the proteins and their hydrochlorides should provide yet another test of our hypothesis. Table V shows data for four such substances.

In the second half of the table, the oxygen and chlorine figures are corrected for the difference in nitrogen content between each protein and its hydrochloride. The chlorine figures are then further adjusted by subtracting the theoretical value of the base-bound chlorine, and the residue is expressed as its oxygen equivalent. This is the amount by which we should expect the oxygen content of the protein to be reduced on conversion to the hydrochloride. The observed decreases in oxygen content of 1·37% for collagen and 1·06% for silk are very near the respective calculated figures of 0·94% and 1·00%. In view of the nature of the problem and the fact that the oxygen figures bear the accumulated errors of each analysis, great accuracy is not claimed for the above calculations. Nevertheless, as each protein is very similar chemically and physically to its hydrochloride, it is reasonable to suppose that some of the errors of analysis will tend to be cancelled out in the figure for the decrease in oxygen content. We therefore consider that these arguments lend strong support to the mechanism we have proposed.

Three lines of evidence have now been adduced. It has been shown that the qualitative and quantitative changes which are observed when a protein combines with dry hydrogen chloride are consistent with the view that two reactions are proceeding simultaneously. Rapid addition resulting in salt formation occurs at the basic groups of the arginine, histidine, lysine, and hydroxylysine residues. A slower substitution reaction proceeds with elimination of water at the aliphatic —OH groups of the serine, threonine, and hydroxylysine side chains.

[The section below cannot be correctly rendered as it contains complex formatting. See the image of the page for a more accurate rendering.]

Table V—Elementary Composition of Proteins and their Hydrochlorides
(Dried in vacuo at 100°)
Collagen Collagen hydrochloride Silk Silk hydrochloride
C (%) 49.29 47.44 48.95 48.45
H (%) 7.16 6.70 6.29 6.42
N (%) 17.70 17.10 18.28 17.28
Cl (%) 5.11 2.39
O (% by difference) 25.85 23.65 26.48 24.86
O (corrected*) 25.85 24.48 26.48 25.42
O difference between protein and hydrochloride 1.37 1.06
Cl (corrected*) 5.29 2.44
Base-bound Cl (theory) 3.21 0.23
Carbon-bound Cl 2.08 2.21
Oxygen equivalent 0.94 1.00

The products of the above reactions, like their parent proteins, can be acetylated. The O-acetyl content of the hydrochloride deriva-

[Footnote] * Calculated to a common nitrogen content with the parent protein.

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tives indicates that some of the —OH groups have been replaced by chlorine.

Finally, elementary analysis of the proteins and their hydro-chlorides shows in the latter a reduced oxygen content of the magnitude predicted by our theory.

Conclusion

The study of the reactions of dry gaseous and ethereal hydrogen chloride with proteins has been extended to silk fibroin and elastin.

Both proteins exhibit similar behaviour to that already described for collagen. They take up relatively large amounts of loosely adsorbed hydrogen chloride, which can be easily removed. Some hydrogen chloride remains firmly combined, with negligible dissociation pressure, even at 100°.

Each protein yields a continuous series of hydrochlorides, the highest containing 2·9% combined HCl for silk and 2·8% for elastin.

Acetylation and elementary analysis of silk fibroin and collagen and their hydrochlorides has confirmed the hypothesis previously advanced to explain the course of the reaction.

The author is indebted to the staff of the Otago University Micro-chemical Laboratory, under the direction of Dr. T. S. Ma, for carrying out the elementary analyses. He also wishes to thank Professor F. G. Soper for permission to use the facilities of his Department, and the Royal Society of New Zealand for a grant from their Research Fund.

References

Blackburn, S., and Phillips, H., 1944. Experiments on the Methylation and Acetylation of Wool, Silk Fibroin, Collagen and Gelatin. Biochem. J., vol. 38, p. 171.

Bowes, J. H., and Kenten, R. H., 1949. Some Observations on the Amino-acid Distribution of Collagen, Elastin and Reticular Tissue from Different Sources. Ibid., vol. 45, p. 281.

Czarnetzky, E. J., and Schmidt, C. L. A., 1934. Studies on the Combination of Certain Amino Acids and Proteins in the Solid State with Certain Gaseous Acids and Bases. J. Biol. Chem., vol. 105, p. 301.

Gordon, A. H., Martin, A. J. P., and Synge, R. L. M., 1943. The Etherification of Hydroxyamino-acid Residues in Silk Fibroin by Dimethyl Sulphate. Biochem. J., vol. 37, p. 638.

Green, R. W., 1948. The Adsorption of Water Vapour on Collagen and Elastin. Trans. Roy. Soc. N.S., vol. 77, p. 24.

—— 1950. The Reaction of Hydrogen Chloride with Dry Proteins. Part 1. Ibid., vol. 78, p. 291.

Hendrix, B. M., and Paquin, F., 1938. The Effect of Alkali Treatment upon Acetyl Proteins. J. Biol. Chem., vol. 124, p. 135.