Hydroxymethylhydroperoxide and Bis(hydroxymethyl)peroxide

and their Effects on Certain Enzymes, Especially Horseradish Peroxidase

AKADEM ISK A V H A N D L IN G

som med benäget tillstånd av Medicinska Fakulteten i Umeå kommer att offentligen försvaras på engelska språket

för vinnande av medicine doktorsgrad i sal A (vån. 2) Lu O, tisdagen den 15 februari kl 10C0

av

STEFAN M ARKLUNDmed. kand.

C entraltryckeriet Umeå, 1972

UMEÅ UNIVERSITY MEDICAL DISSERTATIONSNo. 3 1972

From the Department of Chemistry, Section of Physiological Chemistry, University of Umeå, Umeå, Sweden

Hydroxymethylhydroperoxide and Bis(hydroxymethyl)peroxide

and their Effects on Certain Enzymes, Especially Horseradish Peroxidase

by

STEFAN MARKLUND

Umeå 1972

Centraltryckeriet, Umeå - 1972

This dissertation is based on the following publications :

I. The simultaneous determination of bis(hydroxymethyl)peroxide (BHM P)' hydroxym ethylhydroperoxide (HM P), and H2O2, with titanium 4+. Equilibria between the peroxides and the stabilities of H M P and BHM P at physiological conditions.Acta Chem. Scand., 25 (1971) 3517.

II. Hydroxym ethylhydroperoxide as inhibitor and peroxide substrate of horseradish peroxidase. Eur. J . Biochem. 21 (1971) 349.

III. The actions of hydroxym ethylhydroperoxide and bis(hydroxymethyl)- peroxide on fum arate hydratase, lactate dehydrogenase, aspartate aminotransferase, glucose oxidase, and acid phosphatase. Biochim. Biophys. Acta 258 (1972) 9.

VI. Interactions between hydroxym ethylhydroperoxide and catalase. The rate of formation of the enzyme-substrate compound between hydroxymethylhydroperoxide and horseradish peroxidase.

V. Mechanisms of the irreversible inactivation of horseradish peroxidase caused by hydroxymethylhydroperoxide.

In the following discussion the paper will be referred to by the aboveRoman numerals.

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INTRODUCTION

The reactions between the reducing milieu, maintained by food intake, in living organisms and the ambient oxygen in the form of O2 leads to three types of products.

W ater is formed in the reaction of O2 with the ”end” of the respiratory chain, cytochrome oxidase. This reduction of 0 2 is in general quantitatively the most im portant. Oxygen can also be reduced to water in reactions catalyzed by copper oxidases containing more than two Cu atoms1.

Hydroxylases (monooxygenases) reduce one oxygen atom of O2 to H 20 , whereas the other is introduced into a substrate molecule. O xy­genases (dioxygenases) introduce both atoms of 0 2 into substrates.

Most oxidases (flavoprotein- and copper- containing ^ 2 Cu atoms1) catalyze the reduction of 0 2 to hydrogen peroxide. Under certain con­ditions H2O2 production also may occur in the respiratory chain, possibly via succinate dehydrogenase2. At autooxidation of many compounds, organic as well as inorganic, H2O2 is formed. Thus, metabolically active substances such as ascorbic acid, thiols and hexoses, are powerful peroxide generators under aerobic conditions.

The production of H2O2 at oxidations catalyzed by some flavoprotein oxidases and probably at many other oxidations3-8 occurs by way of the deleterious superoxide radical (0 -2“) which spontaneously forms H2O2 ( 2 0 -2“ + 2H +-> O2 + H2O2). The latter reaction is catalyzed by Cu, Zn or

Abbrevations:

H M P: hydroxym ethylhydroperoxide (HO CH 2OOH )BHM P : bis(hydroxymethyl)peroxide (HOCH 2OOCH2OH)N M R : nuclear magnetic resonance

5

Mn containing enzymes, superoxide dismutases5'6'8'9' which are ubiquit­ously present in aerobic cells and which are suggested to be necessary prerequisites for their survival8 (cerebro-, hepato-, erytro-cuprein in mammals). In water containing 0 2, H2O2 if formed by the action of ionizing radiation, much of it by way of O 2-.10

Unsaturated lipids may be oxidized to noxious lipid peroxides11-13 and this may occur in vitro in e.g. vitamin E deficiency14 and C CI4 poisoning15.

Hydrogen peroxide is damaging to living organisms and must be eliminated. M any systems can accomplish this. Catalases, which are widely distributed in animals, plants and bacteria, rapidly disproportionate H2O2 to O2 and H2O (2H2O2 —> 0 2 + 2H2O). They also catalyze the oxidation of a number of compounds by H2O2 in vitro , but the occurrence of the latter type of reaction in vivo is under discussion. A role for catalase in the oxidation of methanol and/or ethanol is suggested by some results16-18, but is rejected by certain authors19'20.

Peroxidases catalyze the oxidation of a wide variety of substrates by H2O2. They are widespread among plants and occur in some animal cells. Myeloperoxidase in leukocytes apparently contributes to the microbicidal activity of the cells21 and lactoperoxidase in milk, saliva, and tears probably has a similar function22. Thyroid peroxidase seems to effect the iodinations in the thyroid gland23.

Lipid peroxides are not decomposed by catalase or iron-porphyrin peroxidases24, but by glutathione peroxidase, which also catalyzes the reduction of many other peroxides including H2O2 25_28. The enzyme is found in many sites, e.g. liver, heart, erythrocytes, leucocytes, lung, eyes, and kidney, and, unlike catalase and most peroxidases, contains no hematin27. Cytochrome P 450 — 420 catalyze the decomposition of lipid peroxides in vitro29>S0.

H2O2 readily forms addition compounds with many substances, e.g. aldehydes (I)31-33, ketones32'34-36, histidine37-39 and other amino acids40, and carboxylic acid amides40. These addition compounds are often much stronger inactivators of biochemical systems than H2O2 itself (II,

extent to which they are formed in vivo is unknown.

6

PRESENT INVESTIGATION

The present study deals with the organic peroxides which are formed from H2O2 and formaldehyde. They have been stated to impair many im portant biochemical functions. Thus, the peroxide from 2 H C H O and 1 H2O2, bis(hydroxymethyl)peroxide, BHMP, is alleged to be m uta­genic41'42 and to inhibit blood catalase43, anaerobic glycolysis, cell re­spiration, aldolase, glyceraldehyde-3-phosphate-dehydrogenase and Ehrlich ascites carcinoma cells in vitroUA5. A curative effect on the mouse ascites carcinoma in vivo has been reported39. BHM P has been stated to affect cell division, glycolysis and respiration of synchronized yeast cells46 and to inhibit peroxidase51. As peroxidase is known to use as substrates only peroxides containing a free hydroperoxy group, the alleged inhibitory effect of a disubstituted peroxide was challenging.

The effect of BHM P on peroxidase appeared to be complex, and it was soon realized that an investigation of the stability of BHM P in aqueous solution was needed. During the study of agents known to detect H2O2, it was found that in addition to H2O2 both BHM P and H M P (hydroxy- methylhydroperoxide, containing H C H O : H O O H 1:1) gave a yellow colour with titanium 44- in acid aqueous solution. The latter two peroxides reacted more slowly than H2O2 and this was employed in a method for the simultaneous determination of the three peroxides (I). By means of the titanium method it was found that H2O2, H C H O , H M P and BHM P formed an equilibrium. The attainm ent of equilibrium, which was catalyzed by H + and O H -, was very rapid in neutral water solution (I).

The information thus available (I) allotted conditions at which the peroxides were stable enough to permit a study of, and a distinction of their effects on enzymes.

H M P (which can be formed from BHM P by hydrolysis), was found to be an irreversible inhibitor and a prim ary substrate of peroxidase whereas BHM P was completely inactive (II). The influence of substrates on the inhibition reaction was studied, as well as the fate of the hematin group of peroxidase which is the site of attack (V).

Catalase, which is related to peroxidase, could also use HM P as a prim ary substrate, but was much more resistant to the inhibitory action of the peroxide (IV).

7

BHM P (vide s^/?ra)39'41~46'51 and mixtures of H2O2 and H C H O 47-60 have been stated to exert many biochemical effects. Paper III discloses that the effects can be attributed to the formation of HM P, which efficiently inactivates many enzymes.

TH E CHEM ISTRY OF H M P A N D BHM P

Aldehydes and ketones can react with hydroperoxy groups to form a- hydroxyperoxides32.

Ri Ri O O R s

\ \ /C = O + HOORg C

/ / \R2 r 2 o h

(R i = H in aldehydes)

Paper I describes studies on the organic peroxides formed from form ­aldehyde and hydrogen peroxide. The following equilibria are presumed to exist in water solution:

H 2C (O H )2 + H2O2 ^ H O C H 2O O H + H2O (2 )

H 2C (O H )2 + H O C H 2O O H ^ H O CH2O OCH2OH + H2O (3 )

(H 2C(OH)2 = hydrated formaldehyde)

Formaldehyde is almost completely hydrated in water solution Ç> 99.9 °/o)52. However, it is still possible that the formation of H M P and BHM P proceed via free formaldehyde, e.g. instead of (2) we would get:

H2C(OH)2 ^ H C H O + H2O (4)

H C H O + H2O2 3 = H O C H 2O O H (5)

Provided that the reaction in (4) are much more rapid than those in (5), both mechanisms (4, 5 resp. 2) can account for the kinetic and equilibrium results presented in paper I. The reactions in (4)52-54 are in fact faster than the formation of H M P from H2O2 and formaldehyde and the lysis

of HM P, at least below pH ^ 6, depending on the H2O2 concentration.BHM P can be prepared in pure crystalline form, but H M P is always

obtained as an ”oil5’ with H2O2. H M P, BHM P and H C H O (a small amount, not detectable in NM R) in equilibrium (I). Peroxides in addition to H M P and BHM P may be formed by the substitution of OH-groups for H OO -groups55, but were not detectable by N M R in the present peroxide preparations from water solution.

H2O2, H M P and BHM P all give a yellow colour with titanium 4* in acid aqueous solution, though at different rates (BHMP does not actually react with titanium 4+ but forms H M P by acid hydrolysis). The equilibria (formulas 2, 3) may be shifted almost totally towards free H2O2 by the binding of formaldehyde to the amino group56 of Tris in a slightly alkaline Tris buffer. By emploing these findings, a colorimetric method for the simultaneous determination of the three peroxides was developed (I). The method is reasonably rapid ( < 30 min) and has a good precision ( ± 2 % ) . The accuracy seems to be good, as the results agree well with those obtained by analysis with N M R (I).

The equilibrium and rate constants of the reactions in formulas 2 and 3 were determined. H + and O H - catalyze the attainm ent of equilibrium, the effect of OH" predominating at pH > 3. Buffer acid and base also have some catalytic effect. In neutral aqueous solution (pH 7, 25° C) thereactions are rapid; a small amount of H2O2 added to 0.1 M H C H Owill react to 50 % within less than 10 sec, and BHM P hydrolyzes w ith a half time of approx. 30 seconds (I). The activation energy of the OH"- catalyzed hydrolysis of BHM P was also calculated .

The rapidity of the reactions revealed the possibility that biochemical effects ascribed to BHM P39'41-46'51 and found with mixtures of H2O2 and H C H O 47-50 are in fact caused by HM P.

On the basis of the results in paper I, conditions (pH) were selectedat which the hydrolyses of BHM P and H M P were slow enough to permit an analysis of the effects of the peroxides on a number of enzymes.

TH E EFFECTS OF H M P A N D BHM P O N PERO XIDA SE HM P irreversibly inhibited peroxidase. The formerly reported inhibition of peroxidase by BHM P51 could be fully accounted for by the formation of H M P by hydrolysis of BHM P (II).

9

Unlike BHM P, H M P is a prim ary substrate of peroxidase (ki = 2— 5 • 105 sec-1 • M_1, II resp IV), and it probably reacts like H2O257.

k\peroxidase + H2O2 ( ^ ) compound I (6)

k 7compound I + AH2 -> compound II + A H • (7)

compound II + AH2 -> peroxidase + A H • (8)

AH2 = hydrogen donor substrate. Compounds I and II = enzyme-substrate compounds w ith 2 resp. 1 more oxidizing equivalents than Fe3+-peroxidase.

In the presence of 6.7 mM guaiacol (a hydrogen donor substrate) theinactivation reaction between HM P and peroxidase is of the second order with a rate constant of 3.0 • 102 sec-1 • M 1 (II). Variations in the guaiacol concentration (1.7 - 13.3 mM) have very little effect on the rate of the inhibition and this indicates that the reaction does not go by way of HMP-peroxidase-compounds I or II being attacked or undergoing some irreversible change (V).

H2O2 efficiently protects peroxidase against H M P (V). The addition of H2O2 to peroxidase in 6.7 mM guaiacol will transfer much of the peroxidase to compound II (formulas 6, 7, 8) and the protection may be interpretected in terms of compound II being very slightly or not at all sensitive to HM P.

A study of the reaction between peroxidase and H M P as a ”norm al55 prim ary substrate is complicated by the presence of H2O2 in HM P solutions (I) and also by the inhibitory effect of HM P. The rate constant (ki, formula 6) of the reaction was calculated by an indirect method. The amount of guaiacol was determined which could be peroxidized by H M P and a certain amount of peroxidase before all enzyme was inactivated. From this result and the rate constant of the inhibition reaction, ki = 2 • 105 sec-1 • M_1 was obtained (II).

Later (IV) it was found that catalase could remove the H2O2 originally present in a H M P solution as well as that continuously formed by H M P hydrolysis efficiently enough to permit a direct stopped-flow study of the reaction between HM P and peroxidase. A ki of 5 • 105 sec“1 • M“1 was

10

now obtained. The discrepancy between the two results is partly explained by a blocking effect of the guaiacol (6.7 mM)58“59 in the indirect experi­ment. This experiment (II) was also performed with higher H M P con­centrations (50—90 juM) than the stopped-flow runs (6.8— 39 juM) (IV) in which the ki-values showed a tendency to decrease with increasing H M P concentration.

When H M P is added to a peroxidase solution the brown colour changes to emerald green within a few minutes. The green peroxidase compound has a strong absorption band at 670 nm, and its formation is directly correlated to the inhibition of peroxidase by H M P (V). The pyridine hemichrome of the ”670-compound” (V) is essentially identical to that of verdohemochrome60 which is considered to be the last step in heme degradation61, at least in vitro , before biliverdin is formed. A structure with an oxygen bridge instead of a methene bridge has been assigned to it60'62. Upon standing, the ”670-compound” spectrum changes to a bile- pigment type spectrum (V).

However, before the ”670-compound” , a short-lived compound w ith a band at 940 nm appears. It is not formed at conditions giving peroxidase compounds I, II or III, which suggests it to be an intermediate in the inactivation reaction. However, the ”670-compound” does not seem to originate directly from the ”940-compound” (V).

In summary, H M P inactivates peroxidase by attacking the hematin group, the ring of which is finally opened, the minimum sequence being(V):

Free peroxidase + H M P ”940-compound” intermediate -> ”670-compound” —> bile pigment.

Rate constants have been determined for the formation of ”compound I” from a number of hydroperoxides and both peroxidase and catalase.

enzyme peroxide rate constant(sec 1 ■M-1) ref.

peroxidase W iOi 9 10« 63CH 3OOH 1.5 106 63Q H 5O O H 3.6 106 63

catalase H2O2 6 106 64, 65C H 3O O H 8.5 105 66Q H 5O O H 2 104 66

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Increasing size of the hydroperoxide molecule thus leads to a much greater decrease in the reaction rate with catalase than with peroxidase. This indicates that the protein offers more steric hindrance to access to the hematins in catalase than in peroxidase66. H M P reacts, in analogy, faster (approx. 10 times) with peroxidase than with catalase (IV).

The fact that H M P [kt = 2 - 5 • 105 sec-1 • M"1 (II, IV)] reacts more slowly with peroxidase than do ethyl- and methylhydroperoxide is prob­ably not due to a steric effect of the protein but to e.g. hydrogen bonding within the peroxide, solvation of the peroxide or some charge near the hematin.

The large difference in sensitivity to inactivation by H M P between peroxidase (II) and catalase (IV) would be explained by a steric hindrance to the approach of the peroxide to a catalase hematin in an orientation which allows it to oxidize a methene bridge.

Unlike a number of ” thiol-enzymes” (III), peroxide is not (V) pro­tected against H M P by albumin or 2-mercaptoethanol. This indicates that it is less likely that an oxidizing radical in the solution, derived from H M P, inactivates peroxidase. However, it is still possible than an in­activating radical is released from H M P when the peroxide attacks the hematin group.

The inactivation reaction between H M P and peroxidase could possibly take place as a ”side-reaction” in the ”norm al55 process of forming com­pound I from H M P and peroxidase. This would explain why peroxidase- compound II is insensitive or only slightly sensitive to HM P (V).

EFFECTS OF BHM P A N D HM PO N A NUM BER OF N O N -H E M E ENZYMES

Many reports attribute biochemical actions to BHM P (vide supra)‘09'41~46'51, and mixtures of H C H O and H2O2 are known to exert a greater effect on biochemical systems than either substance alone, e.g. as mutagen47"49 or bacteriostaticum50. These investigations were performed w ithout know­ledge of the rapidity at physiological p H of the reactions in formulas 2 and 3 (vide supra). The alleged peroxidase-inhibiting action of BHM P51 was found to be due to H M P formed by hydrolysis (II). Obviously H M P could have produced the other effects of BHM P and H2O2/HCHO

12

mixtures. This possibility was investigated by an examination of the effects of H2O2, H C H O , H M P and BHM P on a number of enzymes (III). O f the above compounds, H M P was by far the strongest enzyme-inhibitor and the slight effect of BHM P could be accounted for by H M P formed by hydrolysis. It was therefore concluded that the alleged effects of BHM P and of mixtures of H C H O and H2O2 could be explained by the formation of HM P.

Fumarate hydratase, lactate dehydrogenase and aspartate amino­transferase, which are ”thiol enzymes” , were most sensitive to HM P. In the case of the latter two enzymes, indirect evidence was obtained that HM P actually attacked essential thiol groups. Glucose oxidase, which is a H2O2 producing enzyme, was quite resistant, but most resistant was potato acid phosphatase. Acid phosphatase from wheat germ is highly radioresistant67.

Albumin and 2-mercaptoethanol protected the ”thiol enzymes” against H M P w ithout reducing the amount of the peroxide. N either did the compounds reactivate the enzymes. Two models that would account for these effects were ventured (III):

a) Some activated species (possibly a radical) derived from H M P attacks the enzymes, w ith albumin or 2-mercaptoethanol acting as scavenger.

b) H M P oxidizes some residue in the enzyme to a state which may either be reversed by albumin or 2-mercaptoethanol or proceed sponta­neously or by further oxidation to an irreversible state.

It would be a special case of a if the active derivative of H M P were produced by the action of a heavy- or transition-metal ion (c.f. Fenton’s reagent)68'69. The protection offered by 2-mercaptoethanol and albumin could then be due to their binding metal ions.

The effects of added FeS0 4 and Cu2+ acetate on the inactivation of fumarate hydratase by H M P were studied59. 1— 100 juM Cu2+ had no effect, whereas 10 jliM Fe2+ increased the rate of inactivation by 30 °/o and 100 /^M Fe2+ by several 100 °/o. However, the inactivation experiments described in paper III were performed with water twice quartz-distilled, and analytical grade reagents which according to specifications would give (Fe) < 0.5 juM in the reaction medium. This concentration of Fe should have a very slight effect on HM P.

13

IN T E R A C T IO N S BETW EEN H M P A N D CATALASE

If catalase is incubated with H M P and samples then are assayed (decline of absorbance at 230 nm of a H202-solution) a decrease in catalatic activity is found (IV). However, during the assay the activity gradually increases and after 1— 2 min the full catalase activity is restored. Catalase is thus reversibly inhibited by H M P, and is much more resistant than peroxidase, which at similar conditions rapidly and irreversibly loses its activity (II).

The visible spectrum of a catalase solution rapidly changes to a compound II type spectrum70 if H M P is added (IV). (No attempts were made to observe an intermediate formation of compound I70.) However, after a while a normal free catalase spectrum returns because added HM P is reduced by ca,talase (vide infra). Peroxidase, when exposed to HM P, is irreversibly transformed into a green compound containing an oxidized hematin group (V). N o such reaction could be observed w ith catalase. A t the conditions of he inhibition experiment (vide supra) almost all catalase is transferred to the enzymatically inactive66 compound II as determined from the increase of absorbance at 435 nm of the catalase solution. (Free catalase and compound I are isobestic at that wavelength70.) Hence the reversal of the inhibition is due to the decay of compound II to free catalase. The ability of H M P59 to induce the formation of catalase compound II is of the same order of magnitude as that of methyl- hydroperoxide66.

When catalase is added to a H M P solution, a disproportionation of the H2O2 actually present occurs, rapid enough to permit a stopped-flow study of the formation of compound I from H M P and peroxidase (IV). H M P is much more slowly attacked by catalase, but the decrease in HM P is markedly accelerated by the addition of hydrogen donor substrates of catalase (IV). Thus, HM P, like H2O2, CH3OOH and Q H 5O O H 66, may serve as a prim ary substrate of catalase. When increasing amounts of hydrogen donor substrate are added, a maximal rate of H M P decrease is approached at which the reaction between H M P and catalase is rate limiting. The ”maximal rate” corresponds to a rate constant for the reaction of 3 • 104 sec-1 • M~4 per mole of hematin (IV).

In the experiments in which H M P appears to serve as a prim ary substrate of peroxidase (II, IV) and catalase (IV), the effect could also be explained by hypothesizing that the free enzymes catalyze the hydrolysis

14

of H M P near the active site and then very efficiently catch the released H2O2 to form compound I, with the hydrolysis as the rate limiting step. It is difficult to distinguish experimentally between this mechanism and a direct reaction between H M P and enzyme. To be operative, however, the indirects mechanism requires that catalase compound I be unable to catalyze the hydrolysis of H M P as this would lead to a catalatic de­composition not only of the H2O2 but also of the H M P in a H M P solution.

GENERAL

H M P inactivated the enzymes mentioned in paper III at least 20 times as rapidly as did H2O2. H M P is very rapidly formed from H2O2 and the aldehyde at physiological p H (I). From the equilibrium constants (formulas 2, 3 above) (I) it can be calculated that 50 % of a small amount of H2O2 will be bound as H M P in 8 mM formaldehyde, and the enzyme destroying effects of H2O2 is expected to be doubled even in 0.4 mM of the aldehyde. One may speculate about the formation of HM P and organic peroxides from other aldehydes, ketones and other compounds (see introduction) in H202-producing biological systems. It has recently been suggested that the formation of aldehydes in myeloperoxidase-H202- CL systems are of importance for antimicrobial activity71.

HM P could possibly be used as a relatively specific inactivator of peroxidase, but the effect of the peroxide on several other types of enzymes, including other hemoproteins remains to be investigated. Catalase is much more resistant (IV) and ” thiol enzymes” , in contrast to peroxidase, can be protected by albumin or 2-mercaptoethanol (III).

As indicated from studies with BHM P39'41-46'51 or mixtures of H2O2 and H C H O 47-50, the effects of which probably arise from H M P (III), the latter peroxide can act as enzyme inhibitor, cytostaticum and mutagen. The higher the pH , the shorter an experiment must be if excessive hydrolysis of H M P is to be avoided (I). At p H > 5.5, equilibrium (formulas 2, 3) is soon reached and most of the peroxide will be in the form of H2O2 if the initial H M P concentration was low. As H C H O will bind to e.g. amino groups56, attention should be given to this kind of effect when working w ith H M P at equilibrium conditions (I).

HM P may prove to be a useful reagent for oxidizable groups in proteins.

15

It is simple to prepare, and can be kept for months at - 20° C. The reaction is easily controlled as the addition of Tris to raise the pH ̂ 8 rapidly removes the H M P (I). The H2O2 that forms can, if necessary, be disproportionated by catalase. However, the reactions of H M P with proteins must be further characterized by amino acid analyses and studies of mechanisms.

GENERAL SUMMARY

1. A novel method is described for the simultaneous determination of H2O2, H M P (hydroxymethylhydroperoxide) and BHM P [bis(hydroxy- methyl)peroxide]. It is based on the different reactivities of the peroxides with titanium 4+ in acid aqueous solution (I).

2. H2O2, H C H O , H M P and BHM P are in equilibrium in water solution. The attainm ent of equilibrium is catalyzed by H + and O H “, predominantly by the latter at pH > 3. In neutral solution all reactions are rapid. The equilibrium constants and the activation energy of the OH~-catalyzed hydrolysis of BHM P are given (I).

3. BHM P, formerly reported to inhibit peroxidase, does not do so, neither does it act as a substrate (II).

4. HM P, which may be formed from BHM P by hydrolysis, irreversibly inactivates free peroxidase in a second-order reaction, k = 3.0 • 102sec_1 • M-1 (II). The enzyme-substrate compound II is much less sensitive or resistant(V).5. HM P inactivates peroxidase by attacking the hematin group, the ring of which is opened by way of at least three intermediates (V).

6. HM P also serves as a prim ary substrate for peroxidase, the rate constant being 2— 5 • 105sec~1 • M“1 (II, IV).

7. Catalase, which reversibly forms the inactive compound II when exposed to HM P, is much more resistant than peroxidase (IV).

16

8. H M P is a prim ary substrate of catalase, k \^>3 • 104sec 1 • M 1 (IV).

9. A number of non-heme enzymes were exposed to H2O2, H C H O , H M P and BHMP. H M P was the strongest inhibitor and the enzymes containing ”essential” thiol groups were most sensitive (III).

10. Biochemical effects attributed BHM P or mixtures of H2O2 and H C H O are probably caused by H M P (III).

11. Albumin and 2-mercaptoethanol protect ” thiol enzymes” against H M P without reducing the amount of peroxide. H M P-inactivated enzymes can not be reactivated by the thiol (III). Albumin and 2-mercaptoethanoi do not proteckt peroxidase against H M P (V).

ACKNOWLEDGEMENTS

First of all I wish to thank Professor Karl-Gustav Paul for suggesting this study, for his continuous, generous, support and guidance and for many stimulating discussions.

I also wish to extend my thanks to Dr. Svante Wold for advice and to Mr. Per-Ingvar Ohlsson for help with many technical problems.

The skilful assistance of Mrs. Rut White and Mrs. Siw öhm an is gratefully acknowledged.

I am also indebted to Mrs. U lla-Britt Mattsson and Miss Mona Arvidsson for typing the manuscripts and to Mr. Sixten Johansson for preparing the illustrations.

I wish to thank Fil. lie. Barbara Steele, Dr. Clarence Loeffler and Mrs. Veronica Bonebrake-Ureland M.A. for linguistic revision.

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