THE REACTION OF IODOACETATE AND OF IODOACETA- … · the reaction of iodoacetate and of iodoaceta- mide with various sulfhydryl groups, with urease, and with yeast preparations* by

THE REACTION OF IODOACETATE AND OF IODOACETA- MIDE WITH VARIOUS SULFHYDRYL GROUPS, WITH

UREASE, AND WITH YEAST PREPARATIONS*

BY C. V. SMYTHE

(From the Laboratories of The Rockefeller Institute for Medical Research, New York)

(Received for publication, April 7, 1936)

It is well established that iodoacetic acid reacts rapidly under physiological conditions, i.e. approximate neutrality and a tem- perature not exceeding 37”, with such sulfhydryl groups as those of cysteine and glutathione (1). It is also known that this re- agent can react with other groups, e.g. amino groups (2), although this reaction proceeds very slowly, if at all, under physiological conditions. However, because of the possibility of the latter re- action, one cannot conclude that reactions inhibited by iodoacetate involve -SH groups. The possibility exists, however, of con- sidering that reactions which are not inhibited by iodoacetate do not involve -SH groups. The only published results, of which the writer is aware, that contradict such a consideration are those of Hellerman, Perkins, and Clark (3) on urease. These authors present a number of experiments which can be interpreted, and perhaps are best interpreted, on the assumption that the activity of urease is dependent on the presence of -SH groups. They found, however, that iodoacetate did not inhibit urease.

In an effort to determine if there are -SH compounds of known structure which either do not react with iodoacetate or at most react very slowly, we have measured the rate of this reac- tion for some typical compounds of this class. The results for thioglucose, thiosalicylic acid, cysteine, glutathione, and thio-

*A preliminary account of these experiments was presented at the Thirtieth annual meeting of the American Society of Biological Chemists at Washington, 1936 (Proc. Am. Sot. Biol. Chem., 8, xcv (1936); J. Biol. Chem., 114 (1936)).

601

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602 Reaction of Iodo Compounds

glycol, at pH 6.1, are given in Fig. 1. For the sake of comparison curves for the reaction of these same compounds with iodoacetam- ide are also given. It can be seen that the rate of the reaction

Minutes FIG. 1. The rate of reaction of iodoacetate and of iodoacetamide with

various sulfhydryl groups at pH 6.1 and 28”. The amount of -SH present was 0.2 cc. of a 0.1 M solution in each case and the amount of iodo compound 0.2 cc. of a 0.5 M solution. The total volume was 1.4 cc. 8 represents thiosalicylic acid and iodoacetamide; X thiosalicylic acid and iodoacetate; A thioglucose and iodoacetamide; A thioglucose and iodoacetate; n cysteine and iodoacetamide; 17 cysteine and iodoacetate; l glutathione and iodoacetamide; 0 glutathione and iodoacetate; 0 thioglycol and iodoacetamide; + thioglycol and iodoacetate.

for these compounds with either iodo compound is in the order given. It is also evident that in each case the iodoacetamide re- acts more rapidly than the iodoacetate. All of these reactions are more rapid at pH 7.1, as can be seen from Table I, which gives

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C. V. Smythe 603

the time required to reach 50 per cent completion. Times of less than 1 minute should be taken to indicate only the order of mag- nitude of the rate, for the reaction was probably more rapid than the establishment of the gas equilibrium being measured.

Ethyl mercaptan does not react with either of these iodo com- pounds at a measurable rate, even at pH 8.3 and 37”. This is an example of a simple -SH compound which gives a good nitro- prusside reaction and is rapidly oxidized by such oxidizing agents as iodine, but which does not react readily with iodoacetate. Phenyl mercaptan reacts quite rapidly, but it is difficult to get

TABLE I Reactions of -SH Compounds with Iodo Con;

-SH compound

Thioglucose . . . . . . . . . . . . .

Thiosalicylic acid. . ‘I I‘

Cysteine. . . . . . . . . ‘I . . . . . . . . . . .

Glutathione.. . . . . ‘I . . . . . .

Thioglycol. . I( . . . . . . . . . . .

Iodo compound

Acetamide Acetate Acetamide Acetate Acetamide Acetate Acetamide Acetate Acetamide Acetate

T 5(

_-

-

pH = 7.1

lkne for I per c*n reaction

%:cf: me i time for iodoace- tamide

min.

0.57 0.81 0.58 0.84 0.81 1.12 1.10 2.0 2.0 7.0

1

t

-

1.42

1.45

1.38

1.82

3.56

wnds

pH = 6.1

Time for 0 per celv reaction

min.

0.7 2.9 1.0 4.0 1.9 5.1 4.5

17.3 17.3 93.5

t

rime for iodosce- the -L time for iodoace- tamide

4.15

4.00

2.68

3.84

5.46

quantitative results with this compound because of its insolubility. Thiourea reacts slowly even at pH 8.3 and 37”. The reaction of the amino groups of urea, cysteine, glutathione, or guanidine with either of the iodo compounds is too slow under these condi- tions to be measured in this way.

The effect of these two iodo compounds on the activity of crystal- line urease and on commercial Arlco urease is shown in Fig. 2. In agreement with the work cited above (3) these curves indicate that urease is very resistant to the action of iodoacetate, but in addition they indicate that it is quite susceptible to the action of

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604 Reaction of Iodo Compounds

iodoacetamide. Probably the greatest factor in this rather large difference is the greater reactivity of iodoacetamide as shown in

Minutes

FIG. 2. The effect of iodoacetate and iodoacetamide on urease activity at 28” and pH 7.0. The experiments were carried out with the usual Warburg manometric apparatus. The main room of the vessel contained 1.00 cc. of NaHCOs of such concentration that the final concentration of NaHCOs was 0.314 M, 0.2 cc. of 10 per cent gum arabic, and the amount of enzyme and inhibitor shown. 15 minutes were allowed to elapse from the time these solutions were mixed until the substrate was added from the side arm and the readings started. Unless otherwise stated 0.2 cc. of 0.1 M urea was always added. The gas room contained 100 per cent CO*. A represents 5 X 10Fmg. of crystalline urease + 0.1 cc. of HzO; n 5 X 10w3mg. of crystal- line urease + 0.1 cc. of 0.1 M ICH&OONa; l 5 X lo+ mg. of crystalline urease + 0.1 cc. of 0.1 M ICH&ONH2; A 1.0 mg. of Arlco urease + 0.2 cc. of HzO; 0 1.0 mg. of Arlco urease + 0.2 cc. of 0.5 M ICH&OONa; 0 1.0 mg. of Arlco urease + 0.2 cc. of 0.5 M ICHICONHS

Fig. 1 and Table I, but the similarity of the amide linkage to that of the enzyme substrate, urea, may also play a role. That the amide linkage alone is not sufficient to account for the effect of

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C. V. Smythe 605

iodoacetamide is evident from the fact that acetamide, although under our conditions it slightly inhibited urease action, had no comparable effect. Cyanacetamide was also non-effective, as was a mixture of acetamide and iodoacetate.

Minutes

FIG. 3. The reaction of iodoacetate and iodoacetamide with crease at 28”. 0 represents 100 mg. of Arlco urease + 0.2 cc. of 1.0 M ICHzCONHz at pH 7.0, total volume 1.20 cc., A 100 mg. of Arlco urease + 0.2 cc. of 1.0 Y ICHGOONa at pH 7.0, total volume 1.20 cc.; X 21.14 mg. of crystalline urease + 0.2 cc. of 0.5 M ICH&ONHz at pH 7.0, total volume 2.20 cc. The activity figures given are in per cent of the initial activity.

If either of these iodo compounds reacts, with the -SH groups of urease, it should be possible to measure this reaction in the same way as with the -SH groups just discussed. Fig. 3 shows the results obtained with each compound acting on 100 mg. of Arlco urease. It is evident that the reaction with iodoacetamide

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606 Reaction of Iodo Compounds

is considerably greater than with iodoacetate. The evidence that the pressure measured here represents a reaction with -SH groups is that the amount of pressure corresponds to the decrease of the nitroprusside reaction. As stated above, the inactivation of the enzyme is also considerably greater with the iodoacetamide than with the iodoacetate, so it makes a consistent picture to assume that this inactivation is due to the destruction of these -SH groups. The possibility still exists, however, that the in- activation is due to the destruction of some other group and is merely accompanied by a destruction of -SH groups, but at the present time we know no such other group.

Fig. 3 also contains a curve for the action of iodoacetamide on crystalline urease (three experiments). It gives a measure of the number of -SH groups destroyed at any time. If the total amount of enzyme is known and one can determine the amount of active enzyme at one or more points along this curve, one can then calculate the number of -SH groups that have been de- stroyed per unit weight of enzyme inactivated. Since it would seem logical to assume that if the inactivation is due to the de- struction of -SH groups one must destroy at least one -SH group per molecule, such data should give a minimum value for the molecular weight of the enzyme. According to Sumner and Poland (4), who estimated the amount of -SH groups from the strength of the nitroprusside reaction, urease contains one -SH group (Le. 32 gm. of sulfur as -SH) per 15,000 gm. This value agrees approximately with the end-value for the iodoacetamide reaction. On this basis one should expect about 30 c.mm. of COZ from the 21.14 mg. of urease used. However, one can see from the curve that the enzyme is 98 per cent destroyed when we have obtained less than half this amount of COZ. Calculated at this point (70 minutes), we have destroyed one -SH group per 40,000 gm. of enzyme inactivated. On the assumption that at least one -SH group is destroyed per molecule, this gives a minimum value of 40,000 for the molecular weight of the enzyme. This value is probably too low, for, as can be seen, the reaction does not stop at this time, but continues. This continuing reac- tion undoubtedly overlaps with the enzyme-destroying reaction. This is well illustrated by the result obtained at 30 minutes. At this point we have already inactivated 73 per cent of the enzyme, but have destroyed only one -SH group per 49,300 gm. of enzyme

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C. V. Smythe

6ooc

0 0 10 20 30 40

607

Minutes

FIG. 4. The effect of iodoacetate and of iodoacetamide on the rate of fermentation by living yeast cells and by cell-free yeast extract at 28”. A represents the living yeast control. The cells were suspended in ~/15 KHtPO,. 0.2 cc. of 40 per cent glucose was added from a side arm at time = 0. The total volume was 1.33 cc. The gas room contained 100 per cent N2. 0 same as the control + 0.03 cc. of 0.1 M ICH&OONa; [7 same as the control + 0.03 cc. of 0.1 M ICH&ONHZ+ A yeast extract control. The main room of the vessel contained 0.5 cc. of Lebedev’s extract + 0.53 cc. of H20. 0.2 cc. of 40 per cent glucose and 0.02 cc. of 0.15 M potassium hexosediphosphate were added from a side arm at time = 0. l same as the control + 0.03 cc. of 0.1 M ICH&OONa; n same as the control + 0.03 cc. of 0.1 Y ICH&ONHz. In each case the iodo compound was added 15 minutes before the substrate was added and the readings started.

inactivated. This value is also probably too low for the minimum molecular weight for the same reason as just given. It would

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608 Reaction of Iodo Compounds

appear then that one can conclude with some assurance that urease can be completely inactivated by iodoacetamide when not more than one-half, and probably not more than one-fourth, of the total -SH groups has been destroyed. If we consider that this inactivation is due to the destruction of the -SH groups and that the destruction of these groups by any other means would also inactivate the enzyme, the above results lead to the following conclusion. If the enzyme is inactivated by oxidizing the -SH groups to -S-S- groups, it cannot be reactivated by HCN, for this reagent yields one -SH group and one -SCN group from each -S-S- group (5). This is a destruction of one-half the -SH groups and this compound will be inactive. The oxidized enzyme can, however, be reactivated by any agent that reduces each -S-S- to two -SH groups. Thus there is no reason to expect that HCN will give the same result as sulfhydryl compounds such as HzS in reactivating this oxidized enzyme. The same may also be true for some other compounds (6).

If the two iodo compounds are compared as inhibitors of fer- mentation by living yeast cells, it is found that the iodoacetate is considerably the better, or more correctly the more rapid, of the two (Fig. 4). The same difference holds for the inhibition of oxygen consumption, with either glucose or ethyl alcohol as sub- strate. This difference is rather surprising in view of the fact that in the -SH reactions tested the difference was in the reverse order, and, since one might expect that if penetration of the cell is to limit the action of either, that this would affect the ionized iodoacetate more than the un-ionized iodoacetamide (7, 8). This permeability effect can be avoided by using cell-free extracts and here also the iodoacetate is the more rapid inhibitor of the two (Fig. 4). It is evident, therefore, that the reaction which causes this inhibition is different from the reaction with any of the -SH groups testred. We know, at present, no grouping that would react rapidly enough under these conditions to account for the iodoacetate effect and yet react more slowly with iodoacetamide.

EXPERIMENTAL

The rate measurements with the iodo compounds and the vari- ous -SH groups were carried out with the usual Warburg mano- metric technique. A sodium bicarbonate-CO2 buffer was used

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C. V. Smythe 609

and the reaction followed by measuring the increase in gas pres- sure due to the CO2 driven out of the liquid by the HI formed from the reaction. 1 molecule of COI: is produced for each molecule of -SH that reacts. 100 per cent CO2 was used in the gas room and the bicarbonate concentration varied to give the pH desired.

The -SH compounds were placed in the main vessel with the

400

d

4

300

Lj 200

100

0 5 10 15

Minutes

FIG. 5. The pressure obtained with different amounts of urea. X represents 0.2 cc. of 0.1 M urea; 0 0.1 cc.‘of 0.1 M urea; 0 0.05 cc. of 0.1 h3 urea. The amount of enzyme was the same in each case.

bicarbonate and the iodo compounds added from a side arm. Controls were run in the same way, without -SH compounds to check any pressure change on mixing and any hydrolysis during the experiment. It was found that the amount of hydrolysis under these conditions was negligible. The same amount of -SH compound (0.2 cc. of 0.1 M solution) was used in every case, so

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610 Reaction of Iodo Compounds

the total pressure to be expected was always the same (448 c.mm.). In general the pressure obtained was slightly less than the theoreti- cal. This is due to the autoxidation of the -SH groups during the preparation of the experiment. This difference, if one works rapidly, is never large, but, it is different for different compounds, so the results are expressed in per cent of the total pressure ob- tained in each case.

The crystalline urease was prepared from jack bean meal according to Sumner (9). The crystals were collected by centri- fuging, washed, and taken up in distilled HzO. The amount of material present was determined by evaporating a portion of the solution to dryness and weighing. The activity of the enzyme was measured manometrically in a bicarbonate-CO2 buffer by measuring the change in the pressure exerted by the gas confined in the vessel. This pressure diminishes during the reaction due to the absorption of COz into the liquid to neutralize the ammonia set free by the reaction. 1 molecule of COZ is absorbed for each molecule of urea split (Fig. 5). The results are expressed in c.mm. of CO2 (1 gm. molecule = 22,400 cc.). 100 per cent COZ was used in the gas room and the bicarbonate concentration was varied to give the pH desired. Readings were made at 1 minute intervals without interrupting the shaking. The urea was always added from a side arm at time = 0.

The error involved in the determination of 1.0 mg. of urea by this method is of the order of 2.0 per cent. For a discussion of the error in the pressure reading see Warburg (10). Further- more, since ammonia is produced during the reaction and the pH is maintained by the sodium bicarbonate-CO2 buffer, a part of the ammonia formed will remain as free ammonia. We can calculate the approximate concentration of free ammonia from the equation

NL+)(OH-> = K

(Am) t.

The (Am) represents the ammonia present as NH3 and as NHkOH. Ka is 1.8 X 10-5. The (OH-) is maintained approximately at lo-’ M. If we use 1.0 mg. of urea in a volume of 1.5 cc. and consider (NHd+) to be approximately equal to the total ammonia formed, we can calculate (Am) to be equal to

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C. V. Smythe 611

(2.2 x 10-~)(10-‘) = 1 2 x 1o-4 M 1.8 X 1O-6 ’

If we express this as c.mm., in keeping with the above, we have 4.0 c.mm. of free ammonia present. This means, of course, that this amount of ammonia has not taken up its equivalent of COZ. The theoretical pressure change to be expected is 374 c.mm., so the error is about 1.1 per cent. The amount of ammonia that will volatilize into the gas room is negligible. From the data given by Bjerrum (11) and that found in Lange (12) one can calculate that under our conditions it will be about 1.0 per cent of the free ammonia present in the solution.

In order that the velocity of the reaction be proportional to the amount of enzyme present it is necessary, since the amount of urea that can be added is limited by the amount of pressure that can be read on the manometer, that one use not more than 0.01 mg. of urease per vessel (total volume of solution about 1.5 cc.). In this dilution in order to get activities as great as those reported by Sumner (9) it is necessary to protect the enzyme from the traces of metal that it is difficult to avoid. The gum arabic recommended by Sumner (13) serves this purpose as does also dinitrosoresorcinol. In order to obtain the readings used in the crystalline urease curve in Fig. 3 it was necessary to use concen- trated enzyme solutions (21.14 mg. in 2.0 cc.). The enzyme activity at different times was obtained by removing a sample from this concentrated solution and diluting to the range where the rate of the reaction is proportional to the amount of enzyme present. The inhibiting effect of the iodo compounds was tested both in the presence of and in the absence of the gum arabic. It has no effect on this reaction. The curves shown in Fig. 3 are corrected for retention of CO, (14).

SUMMARY

The rate at which the following -SH compounds, thioglucose, thiosalicylic acid, cysteine, glutathione, and thioglycol, react with both iodoacetate and iodoacetamide, has been measured. The rates for the various -SH compounds are in the order given, the thioglucose reacting about 30 times as rapidly as the thioglycol (at pH 6.1). In each case the reaction was more rapid with iodoacetamide than with iodoacetate.

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612 Reaction of Iodo Compounds

Ethyl mercaptan does not react at a measurable rate with either iodoacetate or iodoacetamide at pH 8.3 and a temperature of 37”.

The fact that crystalline urease is not readily inhibited by iodoacetate is confirmed. Iodoacetamide is found to be a con- siderably better inhibitor of urease than iodoacetate. The direct reaction of iodoacetamide with urease has been measured. It is concluded that in order completely to inactivate urease by iodo- acetamide it is not necessary to destroy more than one-half and probably not more than one-fourth of the -SH groups present.

In contrast to the relative effect of the two iodo compounds on the other reactions studied, iodoacetate inhibits fermentation by living yeast cells and by cell-free yeast extracts more effectively, or more correctly more rapidly, than does iodoacetamide.

BIBLIOGRAPHY

1. Dickens, F., Biochem. J., 27, 1141 (1933). 2. Michaelis, L., and Schubert, M. P., J. Biol. Chem., 168,331 (1934). 3. Hellermann, L., Perkins, M. E., and Clark, W. M., Proc. Nat. Acad.

SC., 19,855 (1933). 4. Sumner, J. B., and Poland, L. O., Proc. Sot. Exp. Biol. and Med., 30,

553 (1933). 5. Mauthner, J., 2. physiol. Chenz., 78, 28 (1912). 6. Meyer, K., Thompson, R., Palmer, J. W., and Khorazo, D., J. Biol.

Chem., 113, 303 (1936). 7. Goddard, D. R., J. Gen. Physiol., 19,45 (1935). 8. Kohn, H. I., J. Gen. Physiol., 19,23 (1935). 9. Sumner, J. B., J. BioZ. Chem., 69,435 (1926).

10. Warburg, O., Biochem. Z., 162, 51 (1924). Il. Bjerrum, J., K. Dan&e Vidensk. Selsk., Math.-jysik. Medd., 11, 5 (1931). 12. Lange, N. A., Handbook of chemistry, Sandusky (1934). 13. Sumner, J. B., Naturwissenschajten, 16, 145 (1928). 14. Warburg, O., Biochem. Z., 184,481 (1925).

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C. V. SmythePREPARATIONS

WITH UREASE, AND WITH YEAST VARIOUS SULFHYDRYL GROUPS,AND OF IODOACETAMIDE WITH

THE REACTION OF IODOACETATE

1936, 114:601-612.J. Biol. Chem. 

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