Radiation-induced inactivation of enzymes—A review

Radiat. Phys. Chem. Vol. 46, No. I, pp. 123-145, 1995 Copyright © 1995ElsevierScienceLtd 0969-806X(94)00130-8 Printed in Great Britain.All rights reserved 0969-806X/95 $9.50+ 0.00

Pergamon

R A D I A T I O N - I N D U C E D I N A C T I V A T I O N OF E N Z Y M E S - - A REVIEW A. SAHA, P. C. M A N D A L and S. N. BHATTACHARYYA Nuclear Chemistry Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Calcutta 700 064, India (Received I0 July 1994; revised 9 September 1994) Abstract--In vitro studies on the indirect effects of radiation on enzymes have been reviewed. For a better understanding an attempt has been made to classify radiation-induced inactivation of enzymes based on structural features of enzyme molecules. Oxygen effect on enzyme inactivation has also been emphasized. Inactivation caused by reactions with water-derived free radicals as well as with some inorganic and organic free radicals has been discussed.

INTRODUCTION Proteins and enzymes are the core functional units in living systems. Enzymes, in particular, have tremendous roles in biological functions because most of the biological reactions are catalyzed by enzymes. When ionizing radiation, e.g. v-ray, impinges on biological systems, cells are damaged to different extents depending on radiation sensitivity. However, some cellular damages can be restored by some proper repair mechanisms (Simic et aL, 1986). In these repair mechanisms enzymes are also greatly involved. Radiation-induced damages of enzyme molecules can have a pronounced effect on cellular systems because of their loss of biocatalytic properties. Hence, in order to understand cellular damage induced by radiation at the molecular level radiation effects on enzyme molecules constitute a field of increasing interest. In radiotherapy both cancerous and normal cells are damaged, and a determination of the extent of inactivation of enzymes present in the cells will provide information about the cell damage (Stepan et al., 1977; Szeinfeld et al., 1992; Raaphorst et al., 1993). DNA (deoxyribonucleic acid) is usually considered to be the target material on killing of cancerous cells by 7-irradiation. But nowadays cell membranes, or more specifically membrane proteins or enzymes, are also considered as target materials (Wolff et aL, 1986; Verma and Rastogi, 1990). Hence a study of radiation inactivation of enzymes constitutes one of the most important fields of study. It has been established that some processes of aerobic cell metabolism involve the enzymatic production of the superoxide radical O~- and its subsequent removal by superoxide dismutase to yield

hydrogen peroxide and (Fridovich, 1978a, b).

oxygen

[reaction

(1)]

SOD

2 0 I- + 2H20 ~ H202 + 2OH- + 02

(1)

Moreover, there is considerable evidence that a part of the hydrogen peroxide so formed is converted to "OH radicals in vivo in the presence of transition metal ions namely Fe 3+, Cu 2+ by the modified metalcatalysed Haber-Weiss mechanism [reactions (2) and (3)] (Czapski and Ilan, 1978; Koppenol et al., 1978; Halliwell and Gutteridge, 1984). O i - + M "+ ~ 02 + M ("- 1)+

(2)

Mc"- l)+ + H202---, M"+ + OH" + OH -

(3)

The OH" radical can react with most biomolecules very rapidly by a combination of addition, hydrogen abstraction or electron transfer reactions; this makes it an attractive candidate for the cytotoxic action of radical-generating species in vivo. Interaction of reactive oxygen species namely "OH, O1- with proteins or enzymes constitutes one major pathway of their deleterious action in biological environment, because such reactions may cause structural alterations of the protein, e.g. formation or cleavage of disulphide bonds or result in a depletion of enzyme activity by changes within the active sites (Simic et al., 1988; Manning et al., 1989). Hence, in vitro studies of the reactions of radiation generated O1- and "OH radicals with biochemical solutes namely proteins and enzymes can have also useful application in studies on the physiological chemistry of unirradiated biological systems. The use of enzymes in genetic engineering comprises an important area of application of these biomolecules, which perhaps is the fastest growing 123

124

A. Saha et al.

field. The sterilization of enzyme preparations required for the purpose is therefore pertinent and hence may soon become an important practical problem on an industrial scale. The use of radiation in sterilization will depend on the state-of-the-art of free radical chemistry of enzymes. Consequently, a comprehensive understanding of the interactions of free radicals with enzymes is essential for radiation sterilization of enzyme preparations. Radiation sterilization of food will be the major application of radiation in future (Charlesby, 1990). Radiation treatment of protein rich food has to deal with factors which influence the alteration and inactivation of proteins and enzymes. An application of this technology in the food preservation will, therefore, depend on the qualitative and quantitative aspects of radiation chemistry of enzymes. The enzyme inactivation by radiation may be either due to "direct" or "indirect" effect. The study of radiation-induced inactivation of enzymes in the solid state (direct effect) and in dilute aqueous solution (indirect effect) has been directed mainly towards an understanding of events that resulted from the absorption of iomzing radiation in vivo. Radiation inactivation in the solid state is now also widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ (Kempner and Schlegel, 1979; Beauregard et al., 1987; Kempner and Fleisher, 1989). This radiation inactivation method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect "the functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity (Potier et al., 1991). However, till now more emphasis has been given to understand the mechanism of radiationinduced inactivation of a variety of enzyme molecules in aqueous environment. Hence, the focus of this review will be on indirect effects of the primary aqueous radicals on enzyme molecules. In general, although not always, the extent to which enzymes are inactivated by ionizing radiation is related exponentially to the absorbed dose (Adams and Wardman, 1977). Since inactivation arises from reactions of radical products of water radiolysis, the exponential relationship implies that the probability of reaction of a water free radical with an active enzyme molecule is the same as that for reaction with an enzyme molecule that has already been inactivated (Pihl and Sanner, 1963; Buchanan and Armstrong, 1976). However, there are also some cases where inactivation plots are non-exponential. This implies that the reactivity of the site(s) of free radical reaction with the enzyme changes during irradiation, as the enzyme becomes progressively modified by the reaction of free radicals (Nagrani and Bisby, 1989). In the

case of indirect effect, it is well known that radiation inactivation of enzymes is the result of reactions of water-derived radicals with enzyme molecules. Attack by free radicals from water radiolysis can lead to inactivation by preventing both the functions of substrate binding and subsequent catalysis. The prevention of substrate binding and catalytic activity arises due to either conformational changes around the active sites and/or chemical modification of active site residues. The studies of the reactivity of water free radicals which free amino acids show that there must be many sites of reaction in the enzyme, most of which have no relevance to the process of inactivation. For this reason, many inactivation studies have, therefore, been conducted using radicals which react more specifically [e.g. (SCN)2-, Br2-, I2-, N~, CO2-, SeO2- , CC1302, alcohol radicals] (Bisby et al., 1978; Redpath, 1981; Willson, 1982). The selective radicals will attack only one specific amino acid or a group of amino acids. The resulting inactivation of the enzyme can then be attributed to the changes induced in this amino acid or group of amino acids by the specific radicals. Hence, from inactivation studies with these radicals it has been possible to identify tentatively the crucial amino acids of the enzymes, which are responsible for the enzyme activity. A knowledge of the crucial amino acids is necessary to elucidate the radiation-induced inactivation of enzymes. On the other hand, studies on radiation inactivation can provide valuable information regarding the basic properties of enzyme molecules, e.g. amino acid composition of active sites, conformation, etc. There are about two thousand known enzymes. However, properties of many enzymes are still not known in detail. Radiation inactivation studies of enzymes in vitro can therefore bring about a considerable amount of information on the properties of active sites and on the migration of the free-radical function through the macromolecule. Furthermore, the intermediates with which the enzymes may act upon can sometimes be generated either by reduction or oxidation by the selective radicals referred to above and the enzyme functioning can be studied in detail by pulse radiolysis or flash photolysis techniques (Buxton, 1984; Wardman, 1987). These techniques are being applied to study the electron transfer processes involving redox proteins and enzymes (Salmon and Sykes, 1993). No other technique is really found to be appropriate for such studies. REACTIONS OF WATER-DERIVED RADICALS WITH PEPTIDES A N D ENZYMES

The basic structural units of enzymes are amino acids. Many amino acids are joined by peptide bonds to form a polypeptide chain. The chain spontaneously folds on itself in a manner dictated precisely by the sequence of amino acids. The structure is held together by noncovalent bonds, including

Radiation-induced inactivation of enzymes--a review ionic, hydrogen and hydrophobic linkages, and by covalent disulphide bonds. The primary action of 7-rays is described quantitatively by the equation (A) (Burns and Marsh, 1981). 0.429H20 = 0.280e~q 7- 0.297OH + 0.063H + 0.280H + + 0.044H2 + 0.063H202 + 0.003HO 2 (A) Here, the coefficients which are termed as G-values+ are the yields of species in micromoles per Joule of energy absorbed. When enzymes are irradiated in dilute aqueous solution, the reactive species generated in situ due to radiolysis of water react with the constituent amino acids of enzyme molecules. Among the amino acid residues available in biological systems histidine, tyrosine, tryptophan, cysteine and methionine are particularly prone to an attack by reactive water-derived radicals because of the presence of easily oxidizable functional groups. These reactions of water-derived radicals with enzyme molecules lead to enzyme inactivation. Hence, prior to discussing systems comprising radiation-induced enzyme inactivation, a brief survey of the reactions of water-derived radicals with pepfides or enzymes is presented.

125

Reactions with hydroxyl radicals

Hydroxyl radicals react with simple aliphatic amino acids with relatively low rate constants (k ~ 107-108 M -l s -l ) whereas the rate constants are larger (k ~ 101°M - I s -~) with aromatic and sulphur containing amino acids (Butler et al., 1984). In oxygenated solutions the reactions of OH at C - H positions along the protein main chain were found to lead to oxidative degradation via Reactions (4) and (5) followed by hydrolysis of peroxyl radicals to yield P-CONHCH(R)-P + "OH P-CONHC(R)-P + H20 P-CONH(~(R)-P + 02 ---, P-CONHC(O2)R-P

"OH + P-CONHCH(R)-P -- 02 ---, P-CONH2 + RCO-P + HO2

II

I

CH 2

I CH2

CH 2

OH"

I

CH2 ]

~-~; i ~

H

HO~

C

O + H20 + 02

±~ "OH H Reaction (8)

I

~ ~NHCHO

I

CH 2

H

OH

H

Reaction (7)

CH2

O2

I

CH 2

OH

H Reaction (9)

t l G unit-molecules/100eV = 0.1036 #mol/J.

(6)

The attack of "OH via reaction (4) was viewed as occurring in competition with OH attack at side-

RCNHCHR

H

(5)

amide and keto acid functions (Garrison et al., 1962; Atkins et al., 1967; Garrison, 1970). The overall chemistry of this reaction sequence may be represented as follows.

O

+

(4)

CH2

Oil

H

A. Saha et al.

126 RCONHCHR

,~

I

I

I

CH 2

I

CH 2

CH 2

CH 2

+ HO 2 Oil

OH

"O 2

Reaction (10)

Reaction (12)

chain loci, with the yield of main chain degradation being affected by both the amino acid composition and conformational characteristics of the protein. Studies of transient absorption spectra produced by 'OH attack on papain (Clement et al., 1972) and ribonuclease (Lichtin et al., 1972) suggest that 20-30% of the OH' radicals react at C - H positions along the protein main-chain. In the radiolysis of enzymes in aqueous solution the relative amino acid contents and conformations of the protein are such that a major fraction of the "OH radicals are removed through reaction at the more reactive sites, viz., the aromatic and sulphur containing residues. The reactions o f OH" radicals with aromatic amino acids occur mainly at the ring structures (Butler et al., 1984). The radiolytic oxidation of the heterocyclic tryptophan residue in oxygenated solution arises predominantly through reactions initiated by OH addition to unsaturated bonds of the indole moiety as evidenced by both product analysis and pulse radiolysis studies (Armstrong and Swallow, 1969; Winchester and Lynn, 1970). The addition of OH to the C2--C3 double bond of the indole heterocyclic ring, leads to formation of formylkynurenine as a major degradation product in oxygenated solution [reactions (7) and (8)] (Winchester and Lynn, 1970). The addition of OH to the benzenoid ring leads to formation of phenolic products but the yield of this product is relatively low. The pulse radiolysis study using a computerised analysis of the transient absorption spectra suggests that about 40% Of the OH radicals add to the aromatic ring and about 60%o react at the Cz-position [reaction (7)] (Solar, 1985). In oxygen-free solution reaction (9) is suggested to occur. The hydroxycyclohexadienyl radicals formed through the OH addition to the benzene ring of phenyl alanine [reaction (10)] (Brodskaya et al., 1967)

react with oxygen to yield peroxy radical intermediates [reaction (11)] which may undergo the subsequent reactions (12) and (13) to yield tyrosine (ortho, meta and para) as the major products (Brodskaya et al., 1967; Balakrishnan and Reddy, 1970). The radiolytic oxidation of tyrosine residues in oxygen saturated solution appears to involve reactions analogous to those given in reactions (12) and (13), to yield dopa, 3,4-dihydroxy phenylalanine, plus other unidentified products (Fletcher and Okada, 1961; Lynn and Purdie, 1976). Product analysis, ESR (Samuni and Neta, 1973) and pulse radiolysis (Rao et al., 1975; Mittal and Hayon, 1974) studies show that major mechanism for OH attack at histidine residue involves addition to the imidazole ring [reaction (14)]. Among the major products of reaction (14) are asparagine and aspartic acid. One or more of the aforesaid reactions are presumed to occur when these residues are present in enzymes. Aldrich and Cundall adduced the reactions (7-9) to the radiation induced inactivation of lysozyme (Aldrich and Cundall, 1969). Dizdaroglu et al. (1983) have reported the formation of 2-aminobutanoic acid, allo-threonine, o- and m-tyrosine, 3-hydroxytyrosine (dopa) and 2-hydroxytyrosine on irradiation at a dose range of 1 to 8 kGy of lysozyme (10-4M) in oxygen free N20-saturated condition. The formation of these products are explained as follows. 2-aminobutanoic acid is most likely to be an irradiation product of methionine, because it has been identified in the radiolysis of free methionine. The major site of attack of OH on methionine is the sulphur atom; about 80% of OH add to the sulphur atom and 20% abstract hydrogen atoms from the carbon atoms. The strongly electrophilic OH" radical adds on sulphur yielding a sulphuranyl type radical which on elimination of a water molecule gives rise to the formation of a short-lived (ca 200 ns) three-electron bonded [ > S .'. NR2] + type radical cation (Hiller et aL, 1981). It subsequently decarboxylates and the decarboxylation yields depend on the location of the methionine unit in a protein molecule (Bobrowski et al., 1991; Schoneich et al., 1991). The products of OH attack on methionine further predict that cleavage of the C - S - C bond must occur to produce thiyl and alkyl radicals (Gajewski et al., 1984) [reactions (15) and (16)]. 2-aminobutanoic acid, which has been identified as a radiolysis product of methionine-

I

I

d+o =d. CH 2

•

CH 2

OH

"O 2 Reaction (11)

OH

Radiation-induced inactivation of enzymes--a review

T

I

~

T

CH 2

CH 2

],

CH 2

+ H202

+ HO2

OH

"O 2

127

OH

OH

OOH Reaction (13)

O

I

RCNHCHR

I

I

CH 2

,Ly

CH 2

02

N

\ CH /

-O2C OH" +

Products

HO Reaction (14)

H

\ /

~ "O~N 9[

CH 2

CH 2 + S - CH 3

(I)

/ CH

CH2

CH2

H3N+

S - CH 3

\ \ CH /

C H 2 ~ C H 2 - S + CH 3

Reactions (15) and (16) containing proteins, may result from a disproportionation reaction of radical (I). The mechanism of formation of allo-threonine, the diastereoisomer of threonine, is adduced to the production of the following two intermediates (A) and (B) formed due to OH radical attack on threonine in lysozyme. Disproportionation and repair reaction involving A and B by a H" atom transfer may give rise to yield parent L-threonine by retention of configuration and its diastereoisomer allo-threonine by inversion of configuration at the radical carbon atom in the peptide chain. By analogy with reaction (10) the addition of OH radical to the benzene ring of phenylalanine gives the radical (16a), which may lead to the formation of o- and m-tyrosines [reaction (17)]. The formation of dopa and 2-hydroxytyrosine is adduced to OH" radical addition to the aromatic ring at tyrosine to form the radical (17a) which on disproportionation might yield these products.

Since OH radicals react with phenylalanine and tyrosine faster than with threonine, the total G-values of o- and m- tyrosines and hydroxytyrosines in the case of radiolysis of lysozyme are expected to be higher than that of allo-threonine. But the G-value of allo-threonine has been found to be higher than the total G-values of o- and m- tyrosines and hydroxytyrosines (Table 1). The low yields of o- and m-tyrosines and hydroxytyrosines are attributed to sterie factors which play an important role in the reaction of OH" radicals with the particular constituents of lysozyme. High performance liquid chromatography, capillary gas chromatography and mass spectrometry studies have enabled to identify the formation of dimers in the y-radiolysis of phenylalanine and tyrosine peptides in N20-saturated solution (Gordon et al., 1977). The dimer formation in phenyl-peptides occurs by reaction (18). Boguta and Dancewicz (1978, 1981, 1982, 1983) isolated radiation-induced dimers

CH 3

I I

"C

HN

CH

(A) RFC 4611--I

CH 3 OH

CO

¢~

I I

H

C

OH

HN

C

CO

(B)

5

A. Saha et al.

128

CH2

(16a)

I CH

HN

C

II

0

& oH+

I

I

CH 2

I

+ OH

Reaction (17)

+

+ H20

OH

OH

OH

(17a)

CH2 HN

I

CH

c

I

o

of tyrosine and its peptides and attributed them to biphenol type dimers based on their intense fluorescence at around 410 nm [cf. reaction (18)]. There is now definite evidence of similar biphenol type dimer formation during irradiation of enzymes (18a). The dityrosyl cross-link in radiation-induced lysozyme, papain and ribonuclease has been detected through its strong characteristic fluorescence with 2max= 410 nm (Prfitz et al., 1983; Hashimoto et al., 1981, 1982). However, evidence for the formation of the ether type of cross link through C - O - C bonds in OH'-radical induced dimerisation of tyrosine peptides was forwarded for the first time by Karam et al. (1984). But its formation in enzymes is not yet established. Tyrosine phenoxyl radicals have also been found responsible for protein-DNA cross linking (Casas-Finet et al., 1984). The dimers formed may

reduce enzymatic digestibility and contribute to toxicity (Dizdaroglu et al., 1984). It has been suggested from the studies on histone (Deeg et al., 1987) and collagen (Pietrucha and Tubis, 1990) that there is no polymer effect with respect to the yield of the conversion of phenoxyl radicals into dityrosine groups. Although the observed yields of dityrosyl cross-links in these systems are quite low, their detection suggest that similar cross-linking reactions at other sites are also involved but are less readily identifiable. Similar conclusion has also been arrived by determining the yields of dimerisation of ribonuclease, which are found to be 1.3 and 0.85 respectively, initiated by attack of Br~- and OH" radicals (Seki and Schnabel, 1982). The rate of this cross linking process has been followed by pulse radiolysis with light-scattering detection. Values of 2k are of the order of (2-5) x 106 M -1 s -~ for these two initiators. The optical absorption of the TyrO' radical decays with a very similar rate constant. It has been suggested that both Br2- and OH" radicals generate the TyrO" with 70% efficiency, yet the dimer yield differs considerably for these two initiators. Hence, the TyrO" radical cannot be only responsible to account for the observed crosslinking events. OH' radical can abstract H" atom from cysteine residue (PSH) producing PS" radical which can form dimer or in presence of oxygen forms sulphinic (PSOOH) and sulphonic acid derivatives (PSO2 H) [reactions (19)-(21)] (Garrison, 1987).

I

C.H2

~- H2C •

OH

~

Reaction (18)

CH2

+

2H20

poH

Radiation-induced inactivation of enzymes--a review

The reactions of disulphides, cystamine, cystine, etc., with e~q result in formation of the anion radical which absorbs strongly with a band centered at ~410 nm with an extinction coefficient of ~ 104 M -I cm -1.

(18a)

P

HO

RSSR + e~ ~ (RSSR)-

PSH + "OH ~ PS" + H 2 0

(19)

PS" + PS" ~ PS-SP

(20)

PS" + 02 ~ PSO~ --o PSOOH + PSO 2H

(21)

Oxidation of disulphide linkage of cysteine by OH radicals may involve ion-pair formation (Bonifacic et al., 1975). RSSR + "OH ---r (RSSR)- + O H -

(22)

But there is no such report of ion-pair formation with disulphide linkage in enzymes. Although OH" radicals are of strongly oxidizing character, it may lead to reduction of the metal centre in redox proteins. Most of OH" radicals are expected to react with the outer protein coat, and only a small fraction react directly with the metal. Some of these radicals formed on the protein coat seem to transfer an electron intramolecularly to the metal centre, resulting in oxidation of the protein radical and reduction of the metal (Mee, 1987). Reactions with h y d r a t e d electrons

Hydrated electrons react with aromatic amino acids with rate constants of 108109 M-1 s-a (Butler et al., 1984). To illustrate, e~q reacts with phenylalanine to give rise to cyclohexadienyl radical which may undergo dimerization (Mittal and Hayon, 1974). Hydrated electrons react with histidine, cystelne and cystine with rate constants of 109101° M -1 s -1. The reaction of e~q at the cysteine residue (RSH) forms R" radical [reaction (23)] which is in turn removed by H abstraction [Reaction (24)] (Wilkning et al., 1968). RSH + e~q---, R" + SH-

(23)

R" + RSH ----,RS" + RH

(24)

The RS" radical may then dimerise to form disulphide linkage [Reaction (25)] (Rao and Hayon, 1974). 2RS" ~ RSSR

(25)

Table 1. Initial G-values of the products from lysozyme ~-irradiated in N20-saturated aqueous solutions (10 -4 M) Product identified a-amino-n-butyric acid allo-threonine o-tyrosine m-tyrosine 3-hydroxytyrosine 2-hydroxytyrosine (Source: Dizdaroglu et al., 1983.)

G-value (#mol/J) 0.0031 0.0052 0.0010 0.0006 0.0005 0:0008

129

(26)

The first order rate constant for the decay reaction is independent of pH in the range of 4-7.5 but rises sharply with decreasing pH (Hoffman and Hayon, 1972). (RSSR)- ~ RS" -4- RS-

(27)

The chemical consequences of e~q attachment to S-S bonds of enzymes are not fully understood. There appear to be two types of disulphide traps in enzymes (Clement et al., 1972). In one type, the (PSSP)adduct is short lived and undergoes dissociation, as is found with the simple disulphides, cystine, etc. (PSSP)- ---, PS" - PS-

(28)

PS- + H20--* PSH + O H -

(29)

The production of PSH by the reactions (28) and (29) has been observed in aqueous papain (Gaucher et al., 1971). With papain, the dissociation reaction (28) contributes to the overall inactivation yield. The (PSSP)- adducts of the second type are long lived. This long life may be attributed to other intra-molecular forces such as hydrogen bonds which are sufficient to prevent scission. Enzymes that show a high yield of long-lived (PSSP) radicals with little or no short lived component are not significantly inactivated by e~-q. For example, in the pulse radiolysis of both lysozyme (Adams et al., 1969) and ribonuclease (Adams et al.. 1971), neither of which is significantly inactivated by hydrated electrons, the spectra of the electron adducts are long lived and contain no component which decays to any significant extent over the first 20 #s. It has been suggested that the long lived (PSSP)- species are ultimately removed through back reaction with radicals formed through OH attack at various side chain and main chain loci (Ovadia, 1972). Hydrated electrons react with peptides many times faster than with the simple free amino acids due to electrophilicity of peptide bonds. The carbonyl groups of the peptide bond represents a major trapping centre for e~q via reaction (30), in oxygen-free solutions of simple peptides (Rao and Hayon, 1974; Holian and Garrison, 1968) and more complex peptides (Rustigi and Riesz, 1978a, b; Adams et al., 1973). The adducts have weak absorptions in the spectral region below 300 nm and are relatively long lived (Rao and Hyon, 1974). Various types of evidence indicate that the addition of e~q to the peptide carbonyls of enzymes in oxygen-free solutions occurs in competition with e~q addition to the disulphide bond of cystine a n d the protonated histidine. For example, pulse radiolysis studies of papain (Clement

A. Saha et al.

130

O-

OH H20

e;. + RCON.CHR --- R N.CHR2-- R N.C.R2 R e a c t i o n (30)

et al., 1972) and ct-chymotrypsin (Adams et al., 1973)

indicate that [reaction (30)] represents a major path for removal of e ~qin these systems with the remainder being trapped predominantly at cystine disulphide linkages and at histidine residues. Adams et al. (1973) predicted that the amount of electron addition to the disulphide bridges in ~-chymotrypsin could not be more than 1/6 of the total electron yield. On the other hand, with lysozyme and trypsin, most of the hydrated electrons ( ~ 6 0 % ) react with cystine residues (Bisby et al., 1976; Hoffman and Hayon, 1975). R e a c t i o n s with hydrogen a t o m s

It is to be expected that the radicals formed from the reactions of hydrogen atoms with amino acids should be similar to those of either the hydroxyl radicals or the hydrated electron. But due to difficulties of investigating hydrogen atom reactions, relatively little information is available. However, the rate constants with aromatic amino acids ( ~ 1 0 9 M - l s -1) are much higher than that with simple aliphatic amino acids ( ~ 107 M -l s -1) (Butler et al., 1984). The hydrogen atom adduct spectra of lysozyme, ribonuclease and subtilisin novo show absorptions between 300-400 nm (Bisby et al., 1976). It will not be out of place to mention that radical migration is an important characteristic that may be induced in an enzyme molecule by the action of radiation. Spectral changes observed after pulse radiolysis of some enzymes have established that radical migration may occur within the molecule. Consequently, the damage caused by oxidizing radicals in such biological systems may appear at positions different from the initial site of attack. This may result in a change of the native conformation of the protein molecule and in the loss of enzymatic activity (Torchinsky, 1979). In the reaction of H atom, "OH radical, or e~q with ribonuclease, intramolecular radical transfer is clearly established (Shafferman and Stein, 1975). The primary addition of the reactive intermediates to sites on the enzyme molecule is followed by an intramolecular chain of events in which one electron equivalent radical transfer occurs from one amino acid residue to another, involving selectively only divalent sulphur and aromatic residues. This is consistent with the results of steadystate radiolysis showing that these types of amino acid residues are chemically altered. Methionine residues due to the unique combination of hydrophobicity and nucleophilic reactivity provided by the thioether group in the side chain are considered to play a key role in the radical migration processes in peptides and proteins. The role of sulphur peptide function in free radical transfer has, in fact, been studied by Priitz et al. (1989). Pulse radiolysis investigations of peptides (Priitz et al., 1981, 1982, 1983,

1986; Priitz, 1987) and of proteins (Butler et aL, 1982; Prfitz et al., 1985) suggest that a direct contact between two redox centres is prerequisite and the peptide bond does not provide a channel for electron transfer. Studies on metalloproteins (McLendon and Miller, 1985; Axup et al., 1988; Faraggi and Klapper, 1988) and peptide-bridged metal complexes (Isied and Vassilian, 1984; Isied et al., 1985, 1988) reveal that the long-range electron transfer in proteins depends on orientation and separation of two redox centres and also on the intrinsic and thermodynamic factors. The temperature dependence of the rate of electron transfer seems to indicate electron tunnelling between the two redox centres (Bobrowski et al., 1987, 1990; Faraggi et al., 1989), In an investigation of the hydrolytic enzyme papain, the time-resolved radical sequences are interpreted in terms of a similar mechanism of intramolecular radical transfer involving tyrosine and disulphide linkages. Even in simple peptides containing tryptophan a n d tyrosine, intramolecular electron transfer occurs between the two residues (Prfitz and Land, 1979). Recently, long range electron transfer between tyrosine and tryptophan in lysozyme has been investigated by Weinstein et al. (1991). They showed that the first-order rate constant for the intramolecular electron transfer between tyrosine and tryptophan in the enzyme remained unaltered between pH 11 to 6.5 and then increased as the pH was lowered further. This pH dependence suggests that changes in structure or ionization state influence the electron transfer rate in protein and enzymes. Similarly, the radical sequences obtained from pulse radiolysis of alcohol dehydrogenase also indicate charge transfer between tyrosine and tryptophan. It is evident from the above discussion that irradiation of an enzyme in aqueous solution may lead to the formation of a variety of products. Identification of the products which are formed in low yield was a difficult task earlier because of lack of availability of sensitive analytical techniques. Due to major advancement in the development of various analytical tools such as capillary gas chromatography, GC-MS, HPLC, etc., measurement of such products has now been made possible. Thus a study of radiation chemistry of lysozyme in recent years has led to the identification and quantitative determination of a large variety of products that are formed in either large or small yield: DETERMINATION OF R O L E OF WATER RADICALS IN ENZYME INACTIVATION

Variation of pH, presence or absence of oxygen and nitrous oxide and other additives have made it possible to investigate the individual role of hydroxyl

Radiation-induced inactivation of enzymes--a review radical, solvated electron and hydrogen atom as inactivating species in different enzyme systems. If irradiation is carried out in deaerated conditions, all three radicals OH', H', e~q can react with enzymes. But irradiation under N20-saturated condition results in scavenging of e~q with formation of an equivalent amount of OH" radicals [reaction (31)]. H20 + N 2 0 + e~-q----~N2 + OH" + O H -

(31)

Again, t-butanol is commonly used as scavenger of OH' radical [reaction (32)] (CH3)3C-OH + "OH--~ (CH3)2CCH2-OH + H20 (32) in deaerated or N20-saturated solution to determine the role of H' and e~q in enzyme inactivation process. In case of flavoenzyme t-butanol should not be used because t-butanol radical has been found to react with flavin (Ahmad and Armstrong, 1982). In many enzymes inactivation is increased in lower pH in deaerated conditions (Aldrich and Cundall, 1969; Mee et al., 1972). Taking the consideration of stoichiometric conversion of eUq to H atom by reaction (33) e~q + H30 + --~ H" + H20

(33)

the increased inactivation at low pH suggests that H" atom is more efficient as inactivating species compared with e~. A quantitative evaluation of the individual radical contribution has been made by several workers in a number of enzymes (Masuda et al.. 1979; Samuni et al., 1980: Mee et al., 1972). If it is assumed that each of the radical species acts independently of the other with its own characteristic efficiency and radical-radical reactions are neglected, the contribution of each radical to enzyme inactivation will be proportional to the product of the radical concentration and efficiency [equation (B)]. Thus,

131

Table 3. Fractionalefficiencyof aqueous radicalsfor inactivation of ribonuelease pH 2.5 3.0 4.0 5.5 6.5 7.5 Mean

fon

fH

.l~,rq

0.09 0.08 0.06 0.06 0.06 0.07 0.07 = 0.01

0.26 0.21 0.28 ~ a a 0.25 _+ 0.03

a 4 -0.07 0.05 0.06 0.06 + 0.01

~Values considered unreliable. (Source: Mee et al., 1972.)

water-derived radicals for inactivation of ribonuclease (Table 2 and 3). OH" radicals are found to be major inactivating species for many enzymatic systems whereas there are several enzymes which are mainly inactivated by H atoms. The unusual effectiveness of H atoms as inactivating species may be due to either a high degree of selectivity for reaction at a few critical residues or augmentation of the initial damage by intra-enzyme chain reactions (Lichtin et al., 1973). EFFECT OF OXYGEN ON RADIATION INACTIVATION OF ENZYMES

The role of oxygen in radiation biology is of great interest. By far the best radiation sensitizer is oxygen which plays a major role in radiation therapy. But the understanding of the oxygen effect is rather poor not only at the cellular level but even at the macromolecular level. The majority of the reports on the radiationinduced enzyme inactivation presents the radioprotective role of oxygen (Aldrich and Cundall, 1969; Adams e t al., 1971, 1979; Masuda et al., 1979; Dubery et al., 1987). The radioprotective power of oxygen is due to its ability to scavenge H atom and eaq as hydroperoxy (HO~) or superoxide radical, (O~-) by reactions (34) and (35). HO~ and O~- are found to be relatively unreactive for these systems.

(B)

H + 02 --* HO~

(34)

where f is the fractional efficiency of inactivation by the individual radical and gi is the initial radical yield under the actual irradiation conditions. The fractional efficiencies of water free radicals are therefore determined from the G (inactivation) values and respective radical yields under different conditions of irradiation. Taking into account the reactivity of the radicals with enzyme, Mee et al. (1972) made a rigorous evaluation of the inactivation efficiency of

e~q + O2---~02-

(35)

G (inactivation) = E f g i

Table 2. Radical yields ( p m o l / J ) ~ gn

gOH

eaq

pH

N2

N 20

N2

N 20

N2

N 20

2.5 3.0 4.0 5.5 6.5 7.5

0.349 0.326 0.20 0.066 0.057 0.057

0.159 0.098 0.061 0.057 0.057 0.057

0.30 0.30 0.285 0.285 0.285 0.285

0.498 0.554 0,560 0.565 0.565 0.565

0.008 0.026 0.137 0.270 0.280 0.280

0.0 0.0 0.0 0.0 0.0 0.0

ayields calculated in the presence o f 7.3 × 10 s M R N a s e using the rate constants o f hydrated electron reactions. (Collected f r o m M e e et al., 1972.)

To explain oxygen protection in case of radiation inactivation of c~-chymotrypsin, Adams and coworkers (1973) predicted another pathway for oxygen protection, namely, "oxygen fixation process". In this process, oxygen binds with the radical centre produced by reaction of water-derived radicals with enzyme molecules and thereby it prevents radical migration to the potential site(s) of damage in the molecule. The role of molecular oxygen (02) as protectant is also evident in radiation-induced inactivation of flavocytochrome b2 (Bhattacharya et al., in press). More recently, an interesting protective effect of oxygen has been observed in the inactivation of metalloflavoenzyme, dihydroorotate dehydrogenase. From the observed greater radiation-induced damage in the presence of superoxide dismutase (SOD) compared with that in the absence of SOD it has been suggested that this protection of enzyme inactivation is the result of protection of two

132

A. Saha et al.

important components namely flavin and tyrosine residues by O2--mediated repair processes (Saha et aL, 1991a, 1993), The repair of flavin damage is thought to occur via the reaction of OH-induced protein (flavin) radical with O2- and tyrosine damage is expected to be repaired by reaction of 02- with tyrosine phenoxyl radical (TyrO'), TyrOH + OH' ~ TyrO' + H20 H20

TyrO" + 02- - - ~ TyrOH + 02

(36) (37)

This is the first report of protection of radiationinduced flavin damage in an enzyme by O~--mediated repair process. The sensitizing effect of oxygen was observed in radiation inactivation of lactate dehydrogenase and penicillinase (Schuessler and Herget, 1980; Samuni et al., 1980; Yamamoto, 1992). In case of lactate dehydrogenase D37 value (dose required to reduce enzyme activity to 37%) in the absence of oxygen was l l 2 0 G y while it was reduced to 680Gy by the presence of oxygen during irradiation. It has been attributed to the fact that oxygen enhances peptide chain breakage. Samuni et al. (1980) in a study on radiation-induced inactivation of penicillinase, showing a moderate overall protective effect by oxygen with respect to helium-saturated solutions, postulated a double role for oxygen: a protective one, by converting hydrated electrons and hydrogen atoms into unreactive superoxide radicals and a sensitizing one, by enhancing the damage already caused by OH radicals by a factor of 2.6. There are also some reports of enzyme inactivation by O1- (Chuaqui and Petkau, 1987; Davies, 1987; Davies and Delsignore, 1987; Davies et al., 1987a, b). The enzyme activity of catalase, glucose oxidase, glutathione peroxidase and ribonucleotide reductase is inhibited by superoxide ions (Chuaqui and Petkau, 1987; Davies, 1987). A number of studies have been made on the reactions of 02- with catalase which is known to show two different enzymatic functions, catalytic and peroxidatic (Gebicka et al., 1987, 1989; Gebicka and Gebicki, 1990; Metodiewa and Dunford, 1992). There are no changes in the peroxidatic activity of catalase when only superoxide exists in irradiated enzyme system (Gebicka et a l . , 1987). Inhibition of catalytic and peroxidatic activities by superoxide radical in the presence of H202 may be due to reactions (38-40) where active ferric catalase is converted to inactive forms: Catalase + H202 ~ Compound I

(38)

explained by the conversion of the inactive long-lived intermediate, Compound II, to the active ferric eatalase. Recently, spectral studies of intermediate species formed by reaction of 0 2 - radical with bovine liver catalase have been made by Metodiewa and Dunford (1992). Like catalase the reactions of superoxide radical anion with another hemoenzyme lactoperoxidase was followed through the time-courses of absorption changes after pulse radiolysis of oxygen-saturated solution (Gebicka and Gebicki, 1993). They put forward the first spectroscopic evidence for lactoperoxidase compound II formation in the reaction of compound I with superoxide anion and postulated that superoxide anion could bind to the porphyrin edge of Compound II, giving a peripherally-modified derivative, stable u p to observation time of 7 s. However, reduction of the heine iron could not be observed. Samuni et aL (1981) reported an unusual copperinduced sensitization of the biological damage due to superoxide radicals. They investigated the role of O2- in the radiation-induced damage of penicillinase in the presence copper (II) ions. In the absence of Cu(II) ions, H', e~q and OH" contribute toward enzyme inactivation, while 0 2 - does not. Copper (II) had no effect on OH-induced inactivation, but the damage originating from e~q and H" radicals decreased with the addition of copper, presumably due to their trapping by the copper (II) ions (Fig. 1). In 300

x J

200

--

100

--

-x

~ 20

1,-

o

~ o lO-

5 --

---4/ 10-6

10-5

10-4

10-3

[CuSO4] (M)

Compound I + 0 2 - ~ Compound II (inactive) (39) Catalase + 02 ~ Compound III (inactive) (40) Gebicka et al. (1987) put forward an interesting evidence of partial post-irradiation recovery of enzyme activity upon storage at low temperature (4°C) after irradiation in the presence of air. This was

Fig. 1. D M F o f c o p p e r (II) on the r a d i a t i o n response o f

penicillinase: Solutions were saturated with the following gases: N 2 (©); N20 (l--l);air (A); air, 0.1 M formate (O); air, 0.1 M formate, 3 mM H202 ( × ). The DMF represents the ratio between the inactivation rate constant (k) obtained in the presence of copper to that found in the absence of copper, both measured under the same set of experimental conditions. (From Samuni et al., 1981.)

Radiation-induced inactivation of enzymes--a review

133

contrast, with O 2- radicals predominant in the system copper dramatically enhanced the damage. This copper-induced sensitization was further increased in the presence of H202 and enhancement of the damage due to Cu(II) was by two orders of magnitude, as compared with an oxygen enhancement ratio (OER) of 2-3 generally found for molecular oxygen. The enhancement of the damage by copper results from the attack of reducing radical species on copper (II) ions bound to the biomolecule and these proteinCu(II) complex can be reduced by O1- radicals yielding protein-Cu(I) species, which in turn react with H202 to locally form secondary OH" radicals that react, on that site, with the protein impairing its biological function. This was the first observation of "site-specific" biological damage by Cu(II)-catalyzed action of OZ-. Lately, Hasan et al. (1994) has shown that O;causes inactivation of bovine intestinal alkaline phosphatase with an efficiency of 0.014. The relatively high value for G (inactivation)by this species suggests that O~- is selective in its site (or sites) of reaction with the enzyme, though no information regarding the location of such a reaction is available.

residues is evident from the spectrum of TyrO" radical obtained in pulse radiolysis experiments (Clement et al., 1972, 1974). It is calculated that these radicals contribute 47% of all radicals formed due to OH" attack. The inactivation by OH" radical is only 6% which indicates that these phenoxyl radicals do not cause any major damage of the enzyme molecule. In fact, results from other studies provide evidences for Cys-25 as the most sensitive target (Pihl and Sanner, 1963; Lin et al., 1975; Lynn and Louis, 1973; Adams and Redpath, 1974). However, the radical formed by the reaction (41) is also generated by the attack of O 2- at cysteine residue in papain in aerated solution [reaction (42)].

INACTIVATION OF SULPHYDRYL ENZYMES

reconverted to PSH by addition of excess cysteine or other thiols (RSH) by reaction (44). However, dimerisation of the enzyme molecule is unlikely on

Investigation of sulphur-containing biomolecules has a long tradition in radiation biology. Thus inactivation studies of sulphydryl enzymes in aqueous solution turned out to be of particular interest (Armstrong and Buchanan, 1978; yon Sonntag, 1987; Durchschlag and Zipper, 1990). This is because sulphydryl groups play important roles in many biological processes. For example, they are involved in protein subunit association, in membrane structure and in the active sites of certain enzymes. Moreover, there are a number of recent publications in which it has been shown that sulphur-containing amino acids namely methionine play a major role in radical migration processes in peptides and proteins (Bobrowski et aL, 1990). The radiation inactivation yields of papain (Lin et al., 1975) and glyceraldehyde3-phosphate dehydrogenase (Buchanan and Armstrong, 1978), both of which are sulphydryl enzymes, in oxygenated solutions are exceptionally high. For example, G (inactivation) value of papain in air-saturated solution is 0.48 of which 0.35 is repairable on treatment with cysteine (Lin et al., 1975). The "OH radicals are responsible for the non-repairable dam~ige. In papain, which has a single SH group of cysteine residue, only ~ 2 0 % of the OH radicals are involved [reaction (41)] in giving rise to nonrepairable damage (Clement et aL, 1972). PSH -f "OH ---*PS" + H20

(41)

Because of a large number of exposed tyrosine units which are highly reactive towards OH" radicals, majority of the remaining OH" radicals attacks at these residues. The attack of OH' radical at tyrosine

PSH + O~- --* PS" + H O f

(42)

This radical subsequently reacts with oxygen leading to the formation of non-repairable products which are presumed to be sulphinic and sulphonic acid derivatives. According to Pihl and Sanner (1963) the radiation-induced oxidation of protein SH groups by peroxide in air-saturated solutions was attributed to disulphide formation [reaction (43)], which could be 2PSH + H202 ---,PSSP + 2H 20

PSSP + 2RSH --* 2PSH + RSSR

(43)

(44)

steric grounds (Glazer, 1970) and in fact, it is not supported by sedimentation studies of papain after treatment with peroxide (Glazer and Smith, 1961). The growing evidence for the existence of sulphenic acid derivatives (PSOH) of protein (Glazer, 1970), which are probably stabilised by their hydrophobic environment, suggests the following reaction as a probable alternative: PSH + H202---* PSOH + H20

(45)

Repair by cysteine or other thiols may then take place by the following reactions: PSOH + RSH---~ PSSR + H20

(46)

PSSR .s_ RSH ~ PSH + RSSR

(47)

There is little information available for enzyme inactivation with high LET radiations. Inactivation of papain with a variety of charged particle beams has been investigated by Bisby et al. (1984). Calculated G-values for radical yields in deaerated solution of papain are listed in Table 4 and G(inactivation) values of papain under different conditions are shown in Table 5. Studies at the LET range (0.2-t570 eV/nm) showed that the effects of oxygen production as a primary radiolysis product in the densely ionized track structure led to significant yields of superoxide at the higher LET range (Burns et al., 1981a, b). G (total) in argon-saturated solutions rises

A. Saha et al.

134

Table 4, Extrapolated and calculated G-values (#mol/J) for radical yields in deaerated solution applicable to the k~cs for papain at the concentration used in the radiolysis experiments

G(OH) G(efq) 0.249 0.249 37 MeWH 0.202 0.202 10 MeVtH 0.124 0.124 35 MeWHe 0.050 0.050 18 MeV4He 0.041 0.041 6.4 MeV4He 0.020 0.020 46 MeV4N 0.013 0.013 30 MeV2°Ne 0 0 (Collected from Bisby et aL, 1984.) Radiation

6°Coy

G(H') 0.053 0.052 0.047 0.032 0.025 0.012 0.006 0

slightly with increasing L E T because the decrease in free radical yields is c o m p e n s a t e d by the increase in G (H202 + 0 2 - ) at higher LET. T h e values o f G (total) in N 2 0 - s a t u r a t e d c o n d i t i o n show the maxim u m at the same L E T ( ~ 140 e V / n m ) as in the curve for G (repairable) in Fig. 2 due to the m a x i m u m in G (H202) in this region. The v a r i a t i o n o f G (total) with L E T m oxygen saturated solutions shows the initial decrease as a result of the fall in radical yields, followed by a levelling off at L E T higher t h a n 100 e V / n m due to the d o m i n a n c e of the c o n t r i b u t i o n of 0 2- a n d H202 to inactivation at higher LET. A n o t h e r sulphydryl enzyme t h a t has been studied extensively is lactate dehydrogenase (LDH). This is relatively a large enzyme molecule (M.W. 1.4 x 105 D a ) consisting of four subunits. B u c h a n a n a n d A r m s t r o n g (1976) showed t h a t r a d i a t i o n - i n d u c e d inactivation o f this enzyme was due to d a m a g e o f Cys-165 residue which is considered to constitute the active site o f the enzyme b u t n o t directly involved in the binding o f the substrate or coenzyme. T h e altera t i o n o f the kinetic properties of L D H for reaction with p y r u v a t e a n d reduced n i c o t i n a m i d e - a d e n i n e dinucleotide ( N A D H ) due to varying doses of y-irr a d i a t i o n suggested t h a t r a d i a t i o n - i n d u c e d enzyme inactivation was the result of destruction o f active site as well as lowering o f affinity to the substrate (Saito, 1978). In lactate dehydrogenase the toss o f enzymatic activity in oxygenated solutions involves SH o x i -

G(O~- ) 0.0027 0.0030 0.0033 0.0048 0.0057 0.0088 0.0207 0.0280

G(H202) G(O~- + H202) 0.062 0.065 0.070 0.075 0.084 0.087 0.093 0.098 0.10 0.106 0.114 0.123 0.099 0.120 0.096 0.124

d a t i o n a n d its G (inactivation) value (,-~0.012) is, however, low. This low inactivation yield c o m p a r e d with that of p a p a i n a n d glyceryldehyde-3-phosphate dehydrogenase is attributed to the fact that the SH groups o f those enzymes are involved in substrate b i n d i n g a n d are highly nucleophilic in nature. Hence, they are more reactive t h a n the SH groups of lactate dehydrogenase, which do n o t have this function b u t conserve three-dimensional structure t h r o u g h hydrogen b o n d formation. Lactate dehydrogenase is n o t inactivated by 0 2 - ( A r m s t r o n g a n d B u c h a n a n , 1978). In this respect lactate dehydrogenase resembles the b e h a v i o u r of glutamate dehydrogenase ( A b u E1 Failat et al., 1983). Gel filtration o f lactate dehydrogenase irradiated u n d e r deaerated conditions showed t h a t cleavage occurred at specific positions in the peptide chain a n d the smaller fragments did n o t show any enzymatic activity whereas higher molecular weight fragments were f o u n d to be enzymatically active (Schuessler a n d Denkel, 1972; Schuessler et al., 1975). F r o m the recent studies with bovine serum a l b u m i n (Schuessler a n d Schilling, 1984) a n d hemoglobin (Puchala a n d Schuessler, 1993) it is suggested t h a t aminoacyl-proline peptide g r o u p is target for the peptide chain scission a n d this is due to the fact t h a t tertiary amide b o n d s are easier to oxidize t h a n seco n d a r y amide bonds. However, further investigation is still required to establish the specific peptide chain b r e a k i n g process.

Table 5. Inactivation G-values (/~mol/J) for total [G (total)], reparable [G (r)] and non-reparable [G (nr)] inactivation of papain irradiated in aqueous buffer (1 mM) at pH 7.0 Radiation Argon N 20 02 LET (eV/nm) G(t) G(r) G(nr) G(t) G(r) G(nr) G(t) G(r) G(nr) 6°Co~' 0.076 0.046 0.030 0.105 0.065 0.039 0.368 0.233 0.135 0.20 (0.030) (0.040) (0.120) 37 MeWH 0.089 0.062 0.027 0.136 0.104 0.032 ---5.2 (0.027) (0.330) 10 MeWH 0.087 0.066 0.21 0.135 0.109 0.026 0.269 0.213 0.056 13.5 (0.021) (0.026) (0.076) 35 MeV4He 0.095 0.083 0.012 0.155 0.136 0.02 0.213 0.168 0.046 55 (0.012) (0.015) (0.043) 18 MeV4He 0.090 0.082 0.0083 0.138 0.124 0.013 0.138 0.111 0.027 80 (0.010) (0.012) (0.034) 6.4 MeV4He 0.114 0.106 0.0077 0.166 0.153 0.012 0.132 0.117 0.014 140 (0.0070) (0.008) (0.014) 111 MeW4N 0.143 0.134 0.0097 0.113 0.096 0.017 0.135 0.117 0.018 420 46 MeVI4N 0.089 0.080 0.0090 0.149 0.14 0.009 0.135 0.119 0.015 650 (0.0089) (0.009) (0.013) 30 MeV2°Ne 0.104 0.094 0.0090 0.104 0.093 0.010 --0.011 1570 (0.0088) (0.0088) (0.0088) (From Bisby et aL, 1984.)

Radiation-induced inactivation of e n z y m e s - - a review 2.0-

~ r~

(A)

1.0 I--

0.5 -0

(B)

1.5 --

n

1.0 0.5

0

I

1 0 -1

I

10 0

l01

I

I

10 2

10 3

I 10 4

L E T (eV n m -1) Fig. 2. (A) Total inactivation G-values [G (total)] (molecules/100 eV) for papain as a function of LET; (B) Repairable inactivation G-values [G(r)] for papain as a function of LET. Solutions saturated with ( O ) argon, (IS]) N 2 0 . (Bisby et al., 1984.)

Buchanan and Armstrong (1976) reported an important observation that NAD binding to this enzyme specifically protects it against inactivation by gamma-irradiation(Fig. 3). These findings emphasize the stabilization of the enzyme conformation by substrate or coenzyme binding to enzyme molecule: However, it is rather difficult to determine directly the radiation sensitivity of the intermediate ternary complexes in the LDH reaction as it reaches equilibrium rapidly. Saito (1978) made an alternative approach in obtaining information on the stabilization of enzyme conformation in these complexes by determining the radiation sensitivity of the ternary complex, which is composed of a substrate analogue, LDH and a coenzyme or that of an abortive ternary Complex pyruvate-LDH-NAD which is formed at excess pyru[NAD]/[LDH] 80

0

7.5 15.0 22.5 (a) I . . . ~ . . - ~ - - *

tO0

--

80 f"

./

.,,~.1

~" 40 d

(c)

60

(d)

2.4

7= --

vate. Oxamate was used as an analogue compound of pyruvate and the radiation sensitivities of the ternary complexes, oxamate-LDH-NADH and pyruvateLDH-NAD were compared with free LDH and the intermediate binary complexes, LDH-NAD and LDH-NADH (Fig. 4). The experimental results indicated that pyruvate could impart specific protection through the formation of the intermediate complex pyruvate-LDH-NADH and the abortive ternary complex pyruvate-LDH-NAD. The cooperative protection by coenzyme and substrate or its analogue took place on the substrate and coenzyme binding sites. The ternary complex was found to be much more effective for the stabilization of the enzyme molecule rather than the formation of binary complex LDH-NAD or LDH-NADH. Further studies are needed to justify the speculation that the formation of the ternary complexes changes the enzyme conformation in the vicinity of essential cysteine residues and thereby protects this residue. The effects of metal ions on radiosensitivity of LDH had also been investigated. Copper (II) ions sensitized the inactivation of LDH threefold whereas Zn 2+, Fe 2+, Fe3+, Co 2+ and Mn 2+ had no effect (Winstead and McNees, 1974). Metal ions may lead to peptide chain breaking by oxidising protein radicals to dehydropeptides which decompose on mild hydrolysis (Garrison, 1987). The effect of y-radiation on another sulphydryl enzyme NADP-linked malate dehydrogenase (M.W. 2.5 x 105 Da) was investigated from the point of view of industrial application of ? -irradiation in the preservation of food. 7-Radiation alters the biochemical balance in fruit tissue and leads to a delay in the onset of ripening or senescence. Malate dehydrogenase plays an important role in the ripening process of climacteric fruits such as the mango (Krishnamurthy

7.5 15.0 22.5 30.0 (b) l ~e~e* I

60

j(

1.6 0.8 0.25 0.50 0.75

0.25

135

/

- ~ j

...:,,/

.n

FI

A

A

A

20~

~

......o •

o

C,

I

o

O i

°

0.50 0.75 1.00

Fraction o f free e n z y m e Fig. 3. (a) and (b) Plots o f per cent activity after a set irradiation time against [NAD]/[LDH]. (c) and (d) Plots of the effective rate of radical inactivation o f L D H against the fraction of free enzyme. (a) and (c) Air-saturated 8 . 2 x 1 0 - 6 M L D H solutions at pH 8.1, irradiated for 50 min (b) and (d) N20-saturated 8.6 x 10 -6 M L D H solutions at pH 8.1, irradiated for 25 min. (From Buchanan and Armstrong, 1976.)

I

I

I

I

I

2

4

6

8

10

C o n c e n t r a t i o n (x 104 M) Fig. 4. Protection effects of coenzymes, pyruvate and oxamate at 30 krad dose. The per cent residual value of the m a x i m u m reaction rate V after exposure in the existence of pyruvate (©), oxamate (O), N A D (A), N A D H (&), 1.0 x 10 .3 M N A D + pyruvate ([]) and 1.0 x 10 .3 M N A D H + oxamate ( I ) . (Saito, 1978.)

136

A. Saha et al.

and Patwardhan, 1971). The in vivo effect of 7-radiation on this enzyme in mango fruit tissue showed a diminished enzyme activity during ripening. It was reported from the stress response of irradiated mango fruit that enzyme activity of malate dehydrogenase could be used as a measure of the effect of 7 -radiation on fruit ripening (Frylink et al., 1987). The inactivation yield (G-value) of this enzyme m aerated solution was 0.007, and the hydrogen atom and hydrated electron were found to be important in the enzyme inactivation (Dubery et al.. 19871. From the changes in both the kinetic parameters namely Michaelis-Menten constant (Kin) and Maximal velocity (Vmax), which are related by the MichaelisMenten equation (C), where II0 and [S] are the initial velocity Vm.x[S] v0 = - Km + [s]

(c)

and substrate concentration respectively, it has been suggested that enzyme inactivation is not only due to destruction of active site residues cysteine and tryptophan but also due to a general denaturation of the enzyme (Viljoen et al., 1987). Further, the malate dehydrogenase is an allosteric enzyme that exhibits positive cooperative interactions between the malate binding sites (Dubery and Schabort, 1981). The catalytic and regulatory properties of the enzyme are significantly altered by the choice of divalent metal cofactor. With Mg 2+ as cofactor, the enzyme exhibited sigmoid kinetics with positive cooperative interactions between the substrate binding sites. Positive cooperative interactions between the NADP binding sites were also observed with Mn 2+ acting as cofactor. The radiation-induced change in the positive cooperativity to negative cooperativity was observed when Mg 2+ or Mn z+ acted as divalent metal cofactor. The change in conformation seems to affect the allosteric properties of the enzyme as well (Viljoen et al., 1987). Alteration or loss of the regulatory properties of malate dehydrogenase may conceivably be equally important as the loss of the catalytic properties, and may contribute to the distortion of the physiological processes in the mango fruit during the postirradiation period (Dubery et al., 1984). INACTIVATION OF SIMPLE ENZYMES CONSISTING O F ONLY POLYPEPTIDES

Radiation effects on lysozyme, ribonuclease and trypsin have been investigated extensively because of their simple structure containing single polypeptide. In lysozyme, inactivation is primarily due to the attack of OH' radical on Trp-108 which is considered to be the active site (Adams et al., 1969). Recently, a comparative study on the effects of OH" scavengers pentoxifylline, uric acid or thymine on oxidative damage to lysozyme by the hydroxyl radical has been made by Franzini et al. (1993). Increasing concentrations of OH" scavengers gave increasing protection

of lysozyme activity showing that none of the scavengers or their secondary free radicals were toxic. Although all three compounds scavenge OH" with high rate constants, their effects were different. Uric acid and pentoxifylline prevented inactivation as well as aggregation whereas thymine inhibited inactivation but did not prevent aggregation.This difference in behaviour has been attributed to the different yields of reducing radicals formed by reactions of OH" radicals with the scavengers. However, further study is necessary to establish the mechanism involved. In contrast to lysozyme, trypsin is inactivated more efficiently by H" atom and e~q than by OH" radical (Masuda et al., 1979). The inactivation has been attributed to reaction of these radicals with disulphide bonds disrupting the active centre. The attack of e2q on disulphide bonds of cystine was observed by the absorption spectrum of electron-cystine adduct. Though pulse radiolysis studies have shown that ~ 8 0 % of the OH-induced absorption can be attributed to formation of tryptophan radical, the very low inactivation efficiency of OH" radical indicates that tryptophan is not important for enzymatic activity of trypsin (Masuda et al., 1979). An interesting pH effect was observed when radiation-sensitivity of trypsin was found to be increased by nine times on lowering of pH from 7.6 to 1.4 (von Sonntag, 1987). Presumably it is because of changes in the tertiary structure of enzyme molecules at low pH, where e~q is converted to H atom, result in an increased accessibility of the deactivating radicals. Unlike lysozyme, ribonuclease which is devoid of tryptophan is inactivated very efficiently by H" atom. A detailed study by Mee et al. (1972) showed that inactivation efficiency of H" atom is about 0.25 whereas that of OH" or e~q is 0.07. Adams et al. (1969) have shown that the efficiency of H atoms increases with an increase in absorbed radiation dose, suggesting that the sensitive site becomes more exposed due to unfolding of the enzyme molecule with increasing radiation inactivation, as shown by Schuessler et al. (1977). The pulse radiolysis studies have indicated a sequence of consecutive reactions of H' atoms with ribonuclease (Lichtin, 1972, 1973). The inactivation of ribonuclease by high LET radiations clearly demonstrates a decreased inactivation yield due to the reduced free radical yields at high LET compared with low LET (Bisby et al., 1975). However, surprisingly thermal neutrons cause greater inactivation of deoxyribonuclease I (Akaboshi et aL, 1982). If it is assumed that the nuclear reactions J4N(n,p)14C of the enzyme and H(n, 7)D of the surrounding water are the main causes for inactivation, the G (inactivation) values are found to be extremely high (approximately 20.2 for the former and 2.2 for the latter) compared with those by 7-radiations. The reason is still obscure. Alkaline phosphatase is widely distributed in animals and microorganisms. The earlier radiationinduced inactivation studies of this enzyme showed

Radiation-induced inactivation of enzymes--a review OH', O~- and e~q were equivalent in their effects on alkaline phosphatase activity (Lynn and Skinner, 1974). However, the recent investigation shows that H" is the most effÉcient deactivating species among the water-derived free radicals ( f n = 0.04, fon = f e ~ = 0.019) (Hasan et aL, 1994). Kinetic studies of the enzyme have suggested that inactivation is due to general denaturation of the protein, rather than destruction of the substrate binding site. Fractionation of irradiated alkaline phosphatase solutions was carried out using Fast Protein Liquid Chromatography (FPLC) (Fig. 5). The fluorescence emission maximum at 412 nm suggests the presence of enzyme dimers formed through tyrosyl residues. Significantly the use of FPLC in these studies has indicated that the fluorescence is associated with a single, well-resolved peak which is attributed to dialkaline phosphatase and general denaturation which resulted in enzyme inactivation may be due at least in part to the formation o f these tyrosine-linked dimers.

INACTIVATION

OF

METALLOENZYMES

There are also reports of the studies of radiation effects on several metalloenzymes. Carboxypeptidase A is a zinc containing enzyme which has multifunctional properties; it can cleave both ester and peptide linkages. Steady state and pulse radiolysis studies showed that radiation-induced inactivation of this enzyme is the result of damages in tyrosine and tryptophan residues (Roberts, 1973). It has been

70 60 !

I

50 = !

=

¢q

40

~

30

o

--

20

.r~

--

10

I

e~

%

-

•

-

I

II

•'•

i

I

I

¢

I

I

• "0

•

0o0

I I

6-ooo-o~o o o o - ~ 10

12

I I

o6o-o 14

Elution volume (cm 3) Fig. 5. Fractionation using FPLC of N2-saturated solutions of irradiated alkaline phosphatase (10 -5 M), dose= 1.0krads on a Mono Q H/R 5/5 ion exchange column. Fractionation details are given in the text. The insert shows the absorbance ( Q - - O ) at 280 nm and the relative fluorescence emission ( O - - O ) at 412nm (excitation wavelength = 332 nm). (From Hasan et al., 1994.)

137

shown that Br~- inactivates the peptidase activity with greater efficiency compared with esterase activity (Roberts and Bugden, 1975). Superoxide dismutase (SOD) is a well-studied enzyme which contains metal ions in its active site. Eukaryotic superoxide dismutase contains both copper and zinc. The inactivation effÉciency of OH" and H" are only 0.04 and 0.1 respectively (Roberts et al., 1974). The reactions of SOD with radicals derived from the radiolysis of water led to the reduction of Cu(II) in the enzyme (Fielden et al., 1973, 1974; Symonyan and Nalbandyan, 1979) and also the destruction of liganding histidine (Barra et al., 1975). Radiation-induced loss of copper and zinc exhibited initially a linear doseeffect relationship and was less severe than the drop in enzyme activity (Chelak and Petkau, 1981). The concentration dependence of inactivation of irradiated SOD distinguishes it from other enzymatic systems. The inactivation yield in aerated, deaerated or NzO-saturated solution was found to increase exponentially with initial enzyme concentration (Chelak and Petkau, 1981). Moreover, in aerated condition at < 10 #M SOD, the inactivation process continued in the subsequent 72h in concentration dependent fashion. The role of metal ions (Zn 2+ or Co z+ ) in inactivation of bovine carbonic anhydrase has been determined by considering the effects on holoenzyme and metal-free apoenzyme. It has been observed that metal ions have no effect on the inactivation caused by oxidizing radicals whereas inactivation by reducing radicals are protected by these metal ions (Redpath et al., 1975). Yeast alcohol dehydrogenase contains zinc in its active site. The radiation inactivation of this enzyme has been thoroughly investigated (Schimazu and Tappel, 1964; Gorin et al., 1967; Badiello, 1974) where it has been shown that e~ does not contribute to the inactivation, whereas H" atoms are considered primarily responsible for enzyme inactivation. Before the investigation made by Abelidis et al. (1987) on alcohol dehydrogenase, no detailed studies on the effects of ionizing radiation on metal-binding sites in any enzymes were carried out though some metals play essential roles in many enzymes. The release of Zn 2+ from y-irradiated yeast alcohol dehydrogenase has been measured by means of atomic absorption spectrometry. Hydroxyl radicals and hydrogen atoms readily cause zinc release with G-values of 0.013 and 0.011 respectively, whereas hydrated electrons are considered not to contribute to the demetallization process. Thus the efficiency of zinc removal from yeast alcohol dehydrogenase by H" atoms was 0.24. and for OH' radicals was 0.05 (based on GH = 0.047 and Gon -- 0.290), i.e.. the efficiency of H" atoms was five timcs greater than that of OH" radicals. The radiolytically generated secondary radical anions I~-, (SCN)~- and Br~- enhance the rate of zinc release. Evidence is presented that the enzyme is demetallized as a result of free radical reactions at

138

A . S a h a et al.

cysteine and histidine residues. An interesting phenomenon that urate protects alcohol dehydrogenase in the absence of oxygen but strongly sensitizes this enzyme in the presence of oxygen has been observed by Kittridge and Willson (1984). The mechanism of the action of urate-derived peroxyl radical is still unknown. Catalase, a tetrameric ferriheme enzyme, is one of the most extensively studied enzymes. According to Lynn and Raoult (1973) the initial destruction of the heme group in the active site of catalase was related to the radiation-induced loss of enzyme activity and OH" radicals were suggested to be the major inactivating species. Gebicka et al. (1987), however, reported that the lowest catalytic activity was observed in the presence of reductive species (e~q and H'). These species are probably responsible for distortion of the quaternary structure of catalase and hence they have less influence on the peroxidatic activity. From the relatively high activity in the presence of all primary radicals (e~q, H', OH') it has been suggested that reductive species can repair damages produced by oxidizing OH" radicals (Gebicka et al., 1987). Gebicka and Metodiewa (1988) showed from the spectral studies that in contrast to other metalloproteins (van Leeuwan et a I , 1978; Whittburn and Hoffman, 1985), no redox processes in the active site of catalase occurred as a result of OH" or H" interaction. However, recently, the mechanism of the reaction of catalase with e~q, H' and OH" has been studied by pulse radiolysis. Some evidences have been found that eUq and H" react with porphyrin ring of catalase to form z~-radical without reduction of heme iron within investigated time span of 1 s after the pulse. OH" radicals react mainly with the protein moiety of the enzyme (Gebicka and Gebicki, 1990). This differs from the fact that iron (III) hemoproteins are generally reduced to the iron (II) state by the hydrated electron (Hassinoff, 1985). The reason for this difference is not yet clear. Studies were also performed to provide mechanistic insights into the action of the radioprotector drug, 2-mercaptopropionylglycine (MPG) following the radiolysis of the detoxifying enzyme catalase (Wary and Sharan, 1988). This study showed that M P G behaved primarily as a radioprotective drug. However, due to the presence of FeZ+/Fe3+ in the heme groups, under certain conditions (low radiation dose, high catalase concentration) there was circumstantial interaction of Fe2+/Fe 3+ with MPG, resulting in the formation of an unstable catalase Fe2+/Fe3+-MPG chelate/complex. This resulted in the radiosensitizing effect of M P G on catalase. Horseradish peroxidase is another ferrihaem enzyme which catalyses the oxidation of various substrates in the presence of hydrogen peroxide. The radiation inactivation of a crude preparation, consisting of about 20 isoenzymes was studied with emphasis on the charge and molecular size properties (Delinc6e and Radola, 1974a). Thin-layer isoelectric

focusing of the irradiated peroxidase revealed a prevailing dose-dependent shift to components with lower isoelectric points and multiple newly enzymatically active components while gel chromatography showed the formation of aggregates partially retaining activity. OH" radical which is most damaging species is responsible for aggregate formation (Delinc6e and Radola, 1974b). Studies on purified individual isoenzymes which differs in their isoelectric points suggest that radiation-induced changes are the sum of the alterations of the individual isoenzymes (Delinc6e and Radola, 1974c). Pulse radiolysis study on the kinetics of the reactions of e ~qwith horseradish peroxidase showed the reduction of ferric state to ferrous state in the enzyme with a yield of 40% (Gebicka and Gebicki, 1991). The role of metal ions e.g. Mg 2÷ and Co 2+ in the radiosensitivity of enzyme glucose isomerase was also investigated (Bachman et al., 1979). The radiation-induced inactivation was mainly due to reaction of OH" radicals with the enzyme. Mg 2+ affected the inactivation kinetics and increased the radiosensitivity of glucose isomerase, whereas Co 2+ had no effect on the enzyme radiosensitivity. On the basis of Michaelis constant values it was concluded that Mg 2÷ and Co 2÷ bind to different sites of the enzyme, and the mechanism of the enzyme inactivation by these ions is different. INACTIVATION OF FLAVOENZYME AND METALLOFLAVOENZYME

The rate of inactivation of the flavoenzyme Daminoacid oxidase by X-radiation in aqueous solution was reported to be increased when its apoenzyme and coenzyme, F A D were irradiated separately (Dale, 1942). Radiation-induced destruction of the isoalloxazine ring of F A D leads to loss of enzymic activity (Winstead and Moss, 1972). The considerable protective effect of ethanol on inactivation suggests that reactions of hydroxyl radicals are the major cause of inactivation in dilute aqueous solution. Near neutral pH, removal of the coenzyme F A D from amino acid oxidase results in greater inactivation by the attack of selective free radicals Br~- and (SCN)~ (Anderson et al., 1977). From pulse radiolysis studies, this increase is found to be associated with attack on tyrosine and tryptophan residues in the enzyme. Inactivation of both the holoenzyme and apoenzyme by selective free radical attack is found to increase with increasing alkalinity. This is consistent with the attack on tyrosine being of major importance. Flash photolysis studies on some metal-containing flavoenzymes have recently been initiated by Tollin and co-workers (Tollin and Hazzard, 1991). In contrast, little attention has been paid to the study of the inactivation of metalloflavoenzymes by free radicals derived from the radiolysis of water. Such metalloflavoenzymes are particularly important because

Radiation-induced inactivation of enzymes--a review they play important roles in the electron transport processes (Lehninger, 1987). Recently radiation inactivation studies on dihydroorotate dehydrogenase ( D O D H ) (M.W. 1.2 × 10SDa) which is a unique metalloflavoenzyme as it contains flavin mononucleotide ( F M N ) and flavin adenine dinucleotide ( F A D ) in equimolar quantities as well as iron-sulphur centers have been carried out (Saha et al., 1992). The physical mechanism for inactivation of D O D H by characteristic X-rays was investigated by Jawad and Watt (1986). They were mainly concerned with the interaction of X-rays with the iron centre in the enzyme and determined the inactivation cross sections. However, the recent studies on the inactivation of D O D H by 7-irradiation in dilute aqueous solution have shown that H" atom and OH" radical have comparable contributions to enzyme inactivation. Further the inactivation of D O D H by (SCN)2-, Br2- and 12implicated the possible involvement of cysteine and tyroslne residues in the catalytic activity of the enzyme. Changes in the kinetic parameters (Kin and //max) of D O D H due to 7-irradiation suggested that radiation-induced inactivation was the result of modification of the substrate-binding sites and that of the active site residues in the enzyme. Evidence for the

0.15 A

0.10

(a)

0.12 0.06

,~ 0.09 8

< <

0.06 0.02

olo3 I 260

I I 340 400

I 500

I 600

0 700 0.01

0.04

(b)

0.02

-0

0

<1 -0.02 -0.04 I

I

I

I

I

300

400

500

600

700

-0.01

Wavelength (nm) Fig. 6. Absorption spectra of 0.45/~ M dihydroorotate dehydrogenase before (A) and after irradiation (B) with 7-rays under N20-saturated condition at a dose of 13.2 Gy (50% inactivation). (b) Difference spectrum of (B) and (A). (Saha et al., 1992).

139

Table 6. Loss of ravin and tyrosine fluorescencein dihydroorotate dehydrogenase (1.38 #M) followingirradiation in aqueous solution (0.2 M phosphate buffer, pH 6.0) Percentage l o s s Percentageloss of ravin of tyrosine Condition fluorescence/Gy~ fluorescence/Gyb Aerated 0.04 0.21 Argon-saturated 3.02 2.80 Nitrous oxidesaturated 3.20 2.40 Nitrous oxidesaturated +0.1 M KBr -5.10 (Collected from Saha et al., a1991a, ~1993.)

reduction of iron-sulphur centres in the enzyme due to reactions with water free radicals has been put forward from the absorption spectra (Fig. 6) of irradiated D O D H . The peak around 590 nm in the difference spectrum was attributed to the reduction of iron-sulphur centres in the enzyme. This reduction might be the result of reactions of H" atoms and OH" radicals with flavin moiety because Fe 3+ could be reduced to Fe 2+ by H ' atoms and the products formed by reactions of O H ' radicals with free flavin (Saha et aL, 1991b, 1992). The iron-centre reduction could also be due to electron transfer from the radical site at the outer protein coat to the metal centre (Mee, 1987). This aspect needs further investigation. Electrophoretic studies indicated that inactivation was not due to fragmentation or intermolecular crosslinking. The damages on r a v i n and tyrosine residues, two important components for enzyme activity, measured either by fluorescence and/or second-derivative absorption spectrophotometric studies nicely resembled to the radiation-induced inactivation of D O D H (Saha et al., 1991a, 1992, 1993). The fluorescence loss of flavin and tyrosine due to irradiation when measured in the presence of denaturant guanidinium chloride showed that similar to inactivation, H ' and OH" contributed equally to damages of both flavin moiety and tyrosine residues in D O D H (Table 6). Radiation-induced changes in fluorescence spectra (Fig. 7) and the iodide quenching studies revealed that microenvironment around the r a v i n moiety in D O D H was altered due to the reaction of waterderived radicals with the enzyme (Saha et al., 1991a). The partial unfolding of the enzyme molecule by irradiation was also evident from tyrosine fluorescence (Saha et al., 1993).

Table 7. Number of tyrosine residues in dihydroorotate dehydrogenase (1.38/~M) after irradiation at doses for 50% inactivationt Number of Irradiation conditions tyrosine residues Unirradiated 35 Aerated 17 Argon-saturated 5 Nitrous oxide-saturated 4 tDoses for 50% inactivation: 180 Gy under aerated condition;40 Gy for Ar or NzO-saturated solutions. (Saha et al., 1993.)

A. Saha et al.

140 1.000

--

/

acid analysis of the irradiated invertase showed that the aliphatic amino acids were relatively unaffected, whereas tyrosine, phenlalanine, methione and histidine residues were mostly damaged. However, destruction of methionine and histidine residues were probably responsible for radiation-induced enzyme inactivation of external yeast invertase.

6.6 Gy 9.9 Gy 3.3 Gy 0 Gy

.~.

INACTIVATION

0.636

500

525

550

__l

570

Wavelength (nm) Fig. 7. Changes of the fluorescence spectra of dihydroorotate dehydrogenase in aerated 0.2 M phosphate buffer after v-irradiation. Spectra of unirradiated and irradiated enzymes (0.28#M) were taken after excitation at 450nm. (From Saha et al., 1991a.) INACTIVATIONOF GLYCOSYLATEDENZYMES Radiation inactivation studies for a glycosylated enzyme namely external yeast invertase was carried out by Nagrani and Bisby (1989). This enzyme is a glycoprotein in which there are nine mannose-rich oligosaccharide chains attached to asparagine residues of the polypeptide chain, each oligosaccharide chain cOntaining between 26 and 54 mannose residues (Trimble and Maley, 1977). This enzyme contains between 40 and 50% mannose by weight (Chu et al., 1983) and the oligosaccharide chains have been thought to stabilize enzymic activity, promote renaturation, protect against proteolysis and to promote oligomeric structure (Chu et al., 1983, 1985). The observed results on inactivation showed that both OH" and e2q contribute to the radiation inactivation process of the enzyme. The oligosaccharide chains, however, afforded protection towards d%mage by OH', but not by e2q (Nagrani and Bisby, 1989). In both N2 and 02 saturated solutions the destruction of mannose was approximately one-half of that in N20saturated solution, showing that OH" was principally responsible for mannose loss (Table 8). The amino

BY SELECTIVE INORGANIC

RADICALS

Many reports on radiation-induced enzyme inactivation have implicated hydroxyl radicals as responsible for at least part of the inactivation in aqueous solution. However, lack of selectivity of OH" radical reactions with constituent amino acids poses a major problem in elucidating the inactivation mechanisms. More specific techniques are needed to identify the amino acid residues which, if damaged by water-derived radical attack, would lead to enzyme inactivation. In enzyme chemistry, stable chemical reagents which damage certain amino acid residues in the enzyme selectively are often used to identify the crucial residues. However, the finding that inorganic free radical ions ( X ) - ) generated by irradiation of aqueous solutions of halide or pseudo-halide ( X - ) are relatively unreactive towards aliphatic amino acids but show specificity in their reactions with aromatic and sulphur-containing residues provided an alternative useful approach to the identification of residues crucial to the activity of a particular enzyme. The following mechanism has been suggested for the formation of such inorganic free radical anions (Behar et al., 1972): OH" + X - ~ X O H - ~ X" + O H -

(48)

X" + X - ~ X~-

(49)

where X is usually SCN, Br, I, C1. The reactions of these secondary radical anions with amino acids involve an electron transfer from the amino acid (AH) followed by deprotonation to form the amino acid radical: X ; - + AH ---*AH + + 2 X -

(50)

AH + ~,~A" + H +

(51)

It has been found that only methionine, cysteine and the ring-containing amino acids namely tryptophan, tyrosine, histidine and phenylalanine, which are usually seen to play important roles in the active

Table 8. Lossof mannosein externalyeastinvertasefollowingirradiation in aqueous solution (1 mM phosphate, pH 7.0) Percentage loss of mannose Irradiation dose N20 02 N2 O2 saturated (kGy) saturated saturated saturated + 10mM formate 0.67 14 7 7 0 1.34 31 17 16 0 2.02 47 24 24 0 2.69 61 --0 3.36 69 --0 (From Nagrani and Bisby, 1989.)

Radiation-induced inactivation of enzymes--a review

141

Table 9. List of enzymes investigated by the selective free radical technique Enzyme Lysozyme Ribonuclease :t-Chymotrypsm Trypsin Papain Carboxypeptidase Subtilisin Carlsberg & Novo Superoxide dismutase Bovine carbonic anhydrase Yeast alcohol dehydrogenase Pepsin D-amino acid oxidase Lactate dehydrogenase Flavocytochrome b2 Glutamate dehydrogenase Malate dehydrogenase External yeast invertase Dihydroorotate dehydrogenase Alkaline phosphatase

Crucial residues identified

References

Trp His His His Trp, Cys Tyr, Trp His

Adams et al. (1969) Adams et aL (1972) Baverstock et aL (1974) Adams et aL (1973) Adams and Redpath (1974) Roberts (1973) Bisby et al. (1974)

His Trp, Tyr, His His. Cys Trp Tyr Cys Cys, Tyr

Roberts et al. (1974) Redpath et al. (1975) Badiello (1974) Adams et al. (1979) Anderson et al. (1977) Buchanan and Armstrong (1976) Bhattacharya et al. (in press) Abu El Failat et al. (1983) Dubery et aL (1987) Nagrani and Bisby (1989) Saha et al. (1992) Hasan et al. (1994)

Cys, Trp His. Met Cys, Tyr Cys, His

sites of certain enzymes, show appreciable reactivity towards these selective probes. The rates of reaction vary considerably with pH due to the interplay of prototropic equilibria in both the amino acid solutes and their associated free radicals (Adams et al., 1972). In enzyme solutions, the effect of pH on the relative reactivities of a given inorganic radical with constituent amino acids is particularly useful for resolving spectral data from pulse radiolysis. Thus, when combined with inactivation measurements, the spectral data provide direct information on the nature of the amino acids involved in the enzyme function. In the early study with lysozyme, the extreme specificity in the reactions of (CNS)~- with tryptophan at neutral pH was used to show that radiation damage to tryptophan led to the enzyme inactivation (Adams et aL, 1969). The striking similarity of the transient spectrum of the product of reaction between free tryptophan and (CNS)~- with that formed on pulse radiolysis of an N20-saturated solution containing lysozyme and thiocyanate confirmed the kinetic evidence (Adams et aL, 1969; Aldrich et al., 1969) that tryptophan is the site of attack of this radical-anion in lysozyme. The implications of pH effects on the reactivities of these selective radical-anions can be exemplified by studies with ribonuclease (Adams et aL, 1972) which has no cysteine and tyrosine. In the pH region 7-11, the reactivity of free tyrosine and methionine with Br 2- shows a large increase relative to that for histidine. If, therefore, damage to either tyrosine, methionine or both residues were responsible for inactivation of ribonuclease, it might be expected that, over this pH range the sensitivity of the enzyme to inactivation would show a continuous increase. However, between pH 8-10 there is a decrease in the efficiency of inactivation. This suggests that not tyrosine and methionine but histidine damage is important for enzyme inactivation. Thus use of these secondary inorganic free radical ions as specific probes led to the tentative identification of amino

acid residues essential to the enzymatic activity of numerous enzymes (Table 9). However, it is important to mention here that the free radical probe technique may be misleading in certain cases due to subsequent free radical migration from the initial site of attack to the potential site of damage (Butler et al., 1982). ENZYME INACTIVATION BY SELECTIVE ORGANIC RADICALS

Besides these oxidizing radicals, there are also some reducing radicals namely formate and alcohol radicals which are selective in their reactions with amino acid residues in enzymes. The formate radicalanion generated by reaction (52) of hydroxyl radical with formate is a more selective reducing species than hydrated electron because the former is a HCOO

+OH'--~CO~- +H20

(52)

much weaker electron donor than the latter (Wardman, 1989). At neutral pH CO 2- reacts with disulphide bridges with a rate constant of the order of 1 0 9 M - i s - l and with all other amino acids at < 107 M -1 s-!. However, the formate radical is less efficient than e~q as inactivating species and this is consistent with the low reactivity of this species with sites available for reduction compared with e~q (Bisby et al., 1975). The selectivity of reductive attack is utilized in studying conformational changes in enzymes caused by radiation, changes in temperature or by other external factors (Adams and Wardman, 1977). Pulse radiolysis studies have shown that the disulphide bridges in lysozyme are more accessible than those in ribonuclease or ~-chymotrypsin (Bisby et al., 1976): In ribonuclease pulse radiolysis of a dilute N2 O-saturated solution of the enzyme containing formate ion gives rise to a transient spectrum of disulphide radical ion of very weak absorption. However, if the solution is preirradiated with several

142

A. Saha et al.

pulses, the intensity of this absorption measured after a subsequent pulse is much stronger. This is because CO~- reacts with some amino acid residue(s) to produce damage which leads to conformational changes in the enzyme and preirradiation with several pulses make the disulphide bridges more accessible to the CO2- radical. Instead of preirradiating, the rise of temperature of the enzyme solution above 55°C also leads to unmasking of the disulphide bridges as evident from the sharp increase in the absorption of - ( - S S ' - - ) - b a n d produced by reaction of CO2- with disulphide linkages. Alcohol radical is generated by H abstraction by hydroxyl radical [reaction (53)]. The reactions of formate and ethanol radicals with ribonuclease led to inactivation of the enzyme (Schuessler, 1975; CH3CH2OH -I- OH' ~ CH3CHOH + H20 (53) Schuessler et al., 1979). The inactivation efficiency of the formate radicals is much greater than that of the ethanol radicals, because under comparable conditions the D37 was 1360 Gy for formate radicals and 3760 Gy for ethanol radicals. Experiments with 14Clabelled ethanol and formate gave evidence that these organic radicals (R') react with ribonuclease or other proteins leading to covalent crosslinking (Schuessler, 1981): R" + Prot --, Prot" + RH (54) Prot' + R" ---, P r o t - - R

(55)

Prot" + Prot" ---, Prot--Prot

(56)

Once the protein radicals are formed, they can react with each other and aggregate according to the reaction (56). However, Schuessler et al. (1992) based on their recent study with bovine serum albumin has modified the above assumption as instead of hydrogen abstraction alcohol radicals bind to protein molecule producing protein-alCohol' radical [reaction (57)]. These protein radicals can react subsequently with alcohol radicals or can . . . . R" + Prot--,i(Prot--R)"

(57)

produce aggregation. In addition, these protein radical species may become crosslinked to DNA. Further study of the radiation-induced crosslinking reactions using labelled organic molecules may provide a new method for studying conformation of proteins and enzymes in solution. CONCLUDING REMARKS

The studies on inactivation of enzymes have been directed mainly towards the understanding of cellular damage at molecular level and to unveil some important properties of enzymes namely active site composition, conformation, radical migration, function, etc. The role of oxygen on radiation-induced reactions in biological systems is still not well-understood. Both sensitizing and protecting effects of molecular oxygen

on radiation-induced inactivation of enzymes are explained in terms of oxygen fixation. However, the requirements for oxygen fixation are not yet established. Further investigations are necessary to determine the role of different amino acids in oxygen fixation process. Presumably, amino acid sequence and protein conformation play an important role in this process. So, the sequence dependence of oxygen fixation process is an open question. Oxygen protection on radiation-induced enzyme inactivation by O2--mediated repair process has only recently been observed in the case of metalloflavoenzyme, dihydroorotate dehydrogenase. Hence, further studies on such systems are required to understand the mode of i repair mechanism. There are a few investigations on the effects of high LET radiations on enzymes. However, cosmic rays are associated with high LET radiations. To understand the effects of cosmic rays emphasis is currently being given to the study of high LET radiation-induced cellular damages. Hence, this encourages further study on interaction of high LET radiation with enzyme molecules. It will be interesting to see the role of metal ions as well as coenzymes on the high LET radiation-induced enzyme inactivation. The selective inorganic radicals so far discussed above in regard to determination of active site residues are useful for cysteine, methionine and aromatic amino acid residues only. However, there are many aliphatic amino acids which are found to constitute active sites of a number of enzymes. The possibility of spontaneous inactivation at higher pH further limits use of these radical species in number of cases. Hence, search should be continued to find out a variety of more suitable species so that all sorts of active site compositions can be determined. Actually, enzymatic systems and their functions are diverse. Enzyme of similar function may vary in constitution depending on its source. Hence, studies on the effects of radiation on enzymes of similar functions but with different compositions can shed light on structural and functional properties of enzymes. Finally, efforts should be made to find out whether the mechanism of radiation-induced inactivation can be generalised.

REFERENCES

Abelidis S., Moore J. S. and Chakravarty A. (1987) Int. J. Radiat. Biol. 52, 413. Abu E1 Failat R. R., Moore J. S. and Davies J. V. (1983) Radiat. Res. 93, 62. Adams G. E. and Wardman P. (1977) Free Radicals in Biology (Edited by Pryor W. A.), Vol. 3, Ch. 2. Adams G. E., Baverstoek K. F., Cundall R. B. and Redpath J. L. (1973) Radiat. Res. 54, 375. Adams G. E., Bisby R. H., Cundall R. B. and Willson R. L. (1969) Int. J. Radiat. Biol. 16, 333. Adams G. E., Willson R. L., Bisby R. H. and Cundall R. B. (1971) Int. J. Radiat. Biol. 20, 405. Adams G. E., Redpath J. L., Bisby R. H. and Cundall R. B. (1973) J. Chem. Soc. Faraday Trans. L 69, 1608.

Radiation-induced inactivation of enzymes--a review Adams G. E., Bisby R. H., Cundall R. B., Redpath J. L and Willson R. L. (1972) Radiat. Res. 49, 290. Adams G. E., Posener M. L., Bisby R. H., Cundall R. B. and Key J. R. (1979) Int. J. Radiat, Biol. 35, 497. Adams G. E. and Redpath J. L. (1974) Int. J. Radiat. Biol. 25, 129. Ahmad R. and Armstrong D. A. (1982) Biochemistry 21, 5445. Akaboshi M., Kawai K. and Maki H. (1982) Int. J. Radiat. Biol. 42, 99. Aldrich J. E. and Cundall R. B. (1969) Int. J. Radiat. Biol. 16, 343. Anderson R. F., Patel K. B. and Adams G. E. (1977) Int. J. Radiat. Biol. 32, 523. Armstrong D. A. and Buchanan J. D. (1978) Photoehem. Photobiol. 28, 743. Armstrong R. C. and Swallow A. J. (1969) Radiat. Res. 40, 563. Atkins H. L., Bennet-Corniea W. and Garrison W. M. (1967) J. Phys. Chem. 71, 772. Axup A. W., Albin M., Mayo S. L.. Crutchley R. J. and Gray H. B. (1988) J. Am. Chem. Soc. 110, 435. Bachman S., Gebicka L. and Gasyna Z. (1979) Nukleonika 24, 1017. Badiello R., Tamba M. and Quintiliani M. (1974) Int. J. Radiat. Biol. 26, 311. Balakrishnan I. and Reddy M. P. (1970) J. Phys. Chem. 74, 850. Barra D., Bossa F., Calabrese L., Rotilio G., Roberts P. B. and Fielden E. M. (1975) Biochem. Biophys. Res. Commun. 64, 1303. Baverstock K., Cundall R. B., Adams G. E. and Redpath J. L. (1974) lnt. J. Radiat. BioL 26, 39. Beauregard G., Maret A., Salvayre R. and Potier M. (1987) Meth. Biochem. A n a l 32, 313. Behar D., Bevan P. L. T. and Scholes G. J. (1972) J. Phys. Chem. 76, 1537. Bhattacharya D., Saha A. and Mandal P. C., Biochim. Biophys. Acta (in press). Bisby R. H., Cundall R. B. and Burns W. G. (1975) ar. Chem. Soc. Faraday Trans. L 71, 1582. Bisby R. H., Cundall R. B, and Davies A. K. (1978) Photochem. PhotobioL 28, 825. Bisby R. H., Cundall R. B., Sims H. R. and Burns W. G. (1984) lnt. J. Radiat. Biol. 46, 261. Bisby R. H., Redpath J. L., Adams G. E. and Cundall R. B. (1976) J. Chem. Soc. Faraday Trans. L 72, 51. Bobrowski K., Schoenich C., Holcman J. and Asmus K.-D. (1991) J. Chem. Soc. Perkin Trans. 2, 353. Bobrowski K., Wierzchowski K. L., Holcman J. and Ciurak M. (1987) Stud. Biophys. 122; 23. Bobrowski K., Wierzchowski K. L., Holcman J. and Ciurak M. (1990) Int. J. Radiat. Biol. 57, 919. Boguta G. and Dancewicz A. (1978) Stud. Biophys. 73, 149. Boguta G. and Dancewicz A. (1981) lnt. J. Radiat. BioL 39, 163. Boguta G. and Dancewicz A. (1982) Radiat. Phys. Chem. 20, 359. Boguta G. and Dancewicz A. (1983) Int. J. Radiat. Biol. 43, 249. Bonifacic M., Schafer K. M6ckel H. and Asmus K. D. (1975) J. Phys. Chem. 79, 185. Brodskaya G. A., Sharpatyi V. A. and Russ J. (1967) J. Phys, Chem. 41, 583. Buchanan J. D. and Armstrong D. A. (1976) Int. J. Radiat. Biol. 30, 115. Buchanan J. D. and Armstrong D. A. (1978) Int. J. Radiat. Biol. 33, 409. Burns W. G, and Marsh W. R. (1981) J. Chem. Soc. Faraday Trans. L 77, 197. Burns W. G. and Sims H. E. (1981b) J. Chem. Soc. Faraday Trans. L 77, 2803. RPC ~ / 1 ~

143

Burns W. G., May R. and Baverstock K. F. (1981a) Radiat. Res. 86, 1. Butler J., Land E. J. and Swallow A. J. (1984) Radiat. Phys. Chem. 24, 273. Butler J., Land E. J., Priitz W. A. and Swallow A. J. (1982) Biochim. Biophys. Acta 705, 150. Buxton G. V. (1984) Adv. lnorg. Bioinorg. Mech. 3, 131. Casas-Finet J. R., Toulme J. J., Santus R., Butler J., Land E. J. and Swallow A. J. (1984) lnt. J. Radiat. BioL 45, 119. Charlesby A. (1990) Radiat. Phys. Chem. 36, 869. Chelak W. S. and Petkau A. (1981) Biochim. Biophys. Acta 660, 83. Chu F. K., Watorek W. and Maley F. (1983) Arch Biochem. Biophys. 223, 543. Chu F. K., Takese K., Guarino D. and Maley F. (1985) Biochemistry 24, 6185. Chuaqui C. A. and Petkau A. (1987) Radiat. Phys. Chem. 30, 365. Clement J. R., Armstrong D, A., Klassen N. V. and Gillis H. A. (1972) Can. J. Chem. 50, 2833. Clement J. R., Lin W. S., Armstrong D. A., Gaucher G. M., Klassen N. V. and Gillis H. A. (1974) Int. J. Radiat. Biol. 26, 571. Czapski G. and Ilan Y. A. (1978) Photochem. Photobiol. 28, 651. Dale W. M. (1942) Biochem. J. 36, 80. Davies K. J. A. (1987) J. Biol. Chem. 262, 9895. Davies K. J. A. and Delsignore M. E. (1987) J. Biol. Chem. 262, 9902. Davies K. J. A., Delsignore M. E. and Li~ S. W. (1987a) J. Biol. Chem. 262, 9908. Davies K. J. A., Lin S. W. and Pacifici R. E. (1987b) J. Biol. Chem. 262, 9914. Deeg K. J,, Katsikas L. and Schnabel W. (1987) Int. J. Radiat. Biol. 51, 527. Delinc6e H. and Radola B. J. (1974a) Radiat. Res. 50, 9. Delinc6e H. and Radola B. J. (t974b) Radiat. Res. 59, 572. Delinc6e H. and Radola B. J. (1974c) Radiat. Environ. Biophys. 11, 213. Dizdaroglu M., Gajewski E. and Krutzsch H. C. (1983) Int. J. Radiat. Biol. 43, 185. Dizdaroglu M., Gajewski E. and Simic M. G. (1984) Int. J. Radiat. Biol. 45, 283. Dubery I. A. and Schabort J. C. (1981) Biochim. Biophys. Acta 662, 102. Dubery I. A., Viljoen B. and Schabort J. C. (1987) Int. J. Biochem. 19, 831 Dubery I. A , Van Rensbuurg L. J. and Schabort J. C. (1984) Phytochemistry 23, 1383. Durchschlag H. and Zipper P. (1990) Z. Naturforsch. 45e, 645. Faraggi M. and Klapper M. H. (1988) J. Am. Chem. Soc. 110, 5753. Faraggi M., De Felippis M. R. and Klapper M. H. (1989) J. Am. Chem. Soc. 111, 5141. Fielden E. M., Roberts P. B., Bray R. C., Lowe D. J., Mautner G. N., Rotilio G. and Calabrese L. (1974) Biochem. J. 139, 49. Fielden E. M., Roberts P. B., Bray R. C. and Rotilio G. (1973) Biochem. Soc. Trans. 1, 52. Fletcher G. L. and Okada S. (1961) Radiat. Res. 15, 349. Franzini E., SeUak H., Hakim J. and Pasquier C, (1993) Biochim. Biophys Acta 1203, I 1. Fridovich I. (1978a) Science 201, 875. Fridovich I. (1978b) Photochem. Photobiol. 28, 733. Frylink L., Dubery I. A. and Schabort J. C. (1987) Phytochemistry 26, 681. Gajewski E., Dizdaroglu M., Krutzsch H. C. and Simic M. G. (1984) Int. J. Radiat. Biol. 46, 47. Garrison W. M. (1987) Chem. Rev. 87, 381.

144

A. Saha et al.

Garrison W. M., Jayko M. E. and Bennett W. (1962) Radiat. Res. 16, 483. Garrison W. M., Kland-English M., Sokol H. A. and Jayko M. E. M. (1970) J. Phys. Chem. 74, 4506. Gaucher G. M., Mainman B. L., Thomson G. P, and Armstrong D. A. (1971) Radiat. Res. 46, 457. Gebicka L. and Gebicki J. L. (1991) Int. J. Radiat, Biol. 59, 617. Gebicka L. and Gebicki J. L. (1990) Radiat. Phys. Chem. 36, 161. Gebicka L. and Gebicki J. L. (1993) Int. J. Radiat. Biol. 63, 565. Gebicka L. and Metodiewa D. (1988) J. Radioanal. Nucl. Chem. Lett. 127, 253. Gebicka L., Metodiewa D. and Bachman S. (1987) J. Radionanal. Nucl. Chem: 116, 77. Gebicka L., Metodiewa D. and Gebicki J. L. (1989) Int. J. Radiat. Biol. 55, 45. Glazer A. N. (1970) Ann. Rev. Biochem. 39, 101. Glazer A. N. and Smith E. L. (1961) J. Biol. Chem. 236, 2948. Gordon S., Schmidt K. H. and Hart E. L (1977) J. Phys. Chem. 81, 105. Gorin G., Quintiliani M. and Airee S. K. (1967) Radiat. Res. 23, 210. Halliwell B. and Gutteridge J. M. C. (1984) In Oxygen Radicals in Biological Systems (Edited by Packer L.), p. 47. Academic Press, New York. Hasan N. M., McCall P. R., Moore Ji S. and Power D. M. (1994) Radiat. Phys. Chem. 43, 233. Hashimoto S., Kira A., Imamura M. and Masuda T. (1982) Int. J. Radiat. Biol. 41, 303. Hashimoto S., Seki H., Masuda T , Imamura M. and Kondo M. (1981) Int. J. Radiat. Biol. 40, 31. Hassinoff B. B. (1985) Biochim. Biophys. Acta 829, 1. Hiller K.-O., Masloch B., Gobl M. and Asmus K.-D. (1981) J. Am. Chem. Soc. 103, 2734. Hoffman M. Z. and Hayon E. (1972) J. Am. Chem. Soc. 94, 7950. Hoffman M. Z. and Hayon E. (1975) J. Phys. Chem. 79, 1362. Holian J. and Garrison W. M. (1968) J. Phys. Chem. 72, 472 i. lsied S. S. and Vassilian A. (1984) J. Am. Chem. Soc. 106, 1732. Isied S. S., Vassilian A., Magnusson R. H. and Schwarz H. A. (1985) J. Am. Chem. Soc. 107, 7432. Isied S. S., Vassilian A., Wishart J. F., Creutz C., Schwarz H. A. and Sutin N. (1988) J. Am. Chem. Soc. 110, 635. Jawad H. H . a n d Watt D. E. (1986) Int. J. Radiat. Biol. 50, 665. Karam L. R., Dizdaroglu M. and Simic M. G. (1984) Int. J. Radiat. Biol. 46, 715. Kempner E. S. and Fleisher S. (1989) Meth. Enzymol. 172, 410. Kempner E. S. and Schlegel W. (1979)Anal. Biochem. 92, 2. Kittridge K. J. and Willson R. L. (1984) F E B S Lett. 170, 162. Koppenol W. H., Butler J. and van Leeuwen J. W. (1978) Photochem. Photobiol. 28, 655. Krishnamurthy S. and Patwardhan M. V. (1971) Phytochemistry 10, 2577. van Leeuwan J. W., Raap A., Koppenol W. H. and Nauta H. (1978) Biochim~ Biophys. Acta 503, 1. Lehninger A. L. (1987) Principles o f Biochemistry, Ch. 17. Worth Publishers, New York. Lichtin N. N., Ogdan J. and Stein G. (1972) Biochim. Biophys. Acta 276, 124. Lichtin N. N., Ogdan J. and Stein G. (1973) Radiat. Res. 55, 69. Lin W. S., Clement J. R., Gaucher G. M. and Armstrong D. A. (1975) Radiat. Res. 62, 438.

Lynn K. R. and Louis D. (1973) Int. J. Radiat. BioL 23, 477. Lynn K. R. and Purdie J. W. (1976) Int. J. Radiat. Phys. Chem. 8, 685. Lynn K. R. and Raoult A. P. D. (1973) Int. J. Radiat. Biol.

24, 25. Lynn K. R. and Skinner W. J. (1974) Radiat. Res. 57, 358. Manning M. C., Patel K. and Borchardt R. T. (1989) Pharm. Res. 6, 903. Masuda T., Ovadia J. and Grossweiner L. I. (1979) Int. J. Radiat. Biol. 20, 447. Mee L. K. (1987) In Radiation Chemistry: Principle and Applications (Edited by Farhataziz and Rodgers M. A. J.), p. 477. VCH Publishers, New York. Mee L. K., Adelstein S. J. and Stein G. (1972) Radiat. Res. 52, 588. Metodiewa D. and Dunford H. B. (1992) Int~ .I. Radiat. Biol. 62, 543. McLendon G. L. and Miller J. R. (1985) J. Am. Chem. Soc. 107, 7811. Mittal J. P. and Hayon E. J. (1974) J. Phys. Chem. 77, 1629. Nagrani S. and Bisby R. H. (1989) Int. J. Radiat. BioL 55, 191. Ovadia J. (1972) Isr. J. Chem. 10, 1067. Pietrucha K. and Lubis M. (1990) Radiat. Phys. Chem. 36, 155. Pihl A. and Sanner T. (1963) Radiat. Res. 19, 27. Potier M., Thauvette L., Michaud L., Suzanne G. and Beauregard G. (1991) Biochemistry 30, 8151. Priitz W. A. (1987) Radiation Research Vol. 2 (8th Int. Congress Radiat. Res., Edinburgh), (Edited by Fielden J. F.. Hendry J. H. and Scott D.), p. 134. Taylor and Francis. Pr/itz W, A., Butler J. and Land E. J. (1983) Int. J. Radiat. Biol 44, 183. Priitz W. A., Butler J. and Land E. J. (1985) Int. J. Radiat. Biol. 47, 149. Pr/.itz W. A., Butler J., Land E. J. and Swallow A. J (1989) Int. J. Radiat. BioL 55, 539. Pr/itz W. A., Butler J., Land E. J. and Swallow A. (1986) Free Radical Res. Commun. 2, 69. Priitz W. A. and Land E. J. (1979) Int. J. Radiat. Biol. 36, 513. Prfitz W. A., Land E. J. and Sloper R. W. (1981) J. Chem. Soc. Faraday Trans. L 77, 281. Pr/itz W. A., Siebert F., Butler J., Land E. J., Menez A. and Montenay-Garestier T. (1982) Biochim. Biophys. Acta. 705, 139. Puchala M. and Schuessler H. (1993) lnt. J. Radiat. Biol. 64, 149. Raaphorst G. P., Feely M. M., Chu G. L. and Dewey W. C. (1993) Radiat. Res. 134, 331. Rao P. S., Simic M. and Hayon E. J. (1975) J. Phys. Chem. 79, 1260. Rao P. S. and Hayon E. J. (1974) J. Phys. Chem. 78, 1193. Redpath J. L. (1981) J. Chem. Ed. 58, 131. Redpath J. L., Santus R., Ovadia J. and Grossweiner L. I. (1975) Int. J. Radiat. Biol. 28, 243. Roberts P. B. (1973) lnt. J. Radiat. Biol. 24, 143. Roberts P. B. and Bugden R. D. (1975) lnt. J. Radiat. Biol. 28, 485. Roberts P. B., Fielden E. M., Rotilio G., Calabrese L., Bannister J. V. and Bannister W. H. (1974) Radiat. Res. 60, 441. Rustgi S. and Riesz P. (1978a) Int. J. Radiat. BioL 34, 127. Rustgi S. and Riesz P. (1978b) Int. J. Radiat. Biol. 34, 449. Saha A., Mandal P. C. and Bhattacharyya S. N. (1991a) Int. J. Radiat. Biol. 60, 769. Saha A., Mandal P. C. and Bhattacharyya S. N. (1991b) Bull. Chem. Soc. Jap. 64, 2532. Saha A., Mandal P. C. and Bhattacharyya S. N. (1992) Radiat. Res. 132, 7. Saha A., Mandal P. C. and Bhattacharyya S. N. (1993) lnt. J. Radiat. Biol. 63, 557.

Radiation-induced inactivation of enzymes--a review Saito M. (1978) Int. J. Radiat. Biol, 34, 95. Salmon G. A. and Sykes A. G. (1993) Meth. Enzymol. 227, 522. Samuni A., Kalkstein and Czapski G. (1980) Radiat. Res. 82, 65. Samuni A., Chevion M. and Czapski G. (1981) J. BioL Chem. 256, 12632. Samuni A. and Neta P. (1973) J. Phys. Chem. 77, 1629. Schimazu F. and Tappel A. L. (1964) Radiat. Res. 23, 210. Sch6neich C., Bobrowski K., Holcman J. and Asmus K,-D. (1991) In Oxidative Damage and Repair: Chemical, Biological and Medical Aspects (Edited by Davies K. E. J.), p. 380. Pergamon Press, Oxford. Schuessler H. (1975) lnt. J. Radiat. BioL 27, 171. Schuessler H. (1981) lnt. J. Radiat. BioL 40, 483. Schuessler H. and Denkel P. (1972) lnt. J. Radiat. Biol. 21, 435. Schuessler H. and Herget A. (1980) lnt. J. Radiat. Biol. 17, 71. Schuessler H. and Schilling K. (1984) lnt. J. Radiat. BioL 45, 267. Schuessler H., Davies J. V. and Herget A. (1979) Studies Phys. Theoret. Chem. 6, 161. Schuessler H., Ebert M. and Davies J. V. (1977) Int. J. Radiat. BioL 32, 391. Schuessler H., Niemczyk P., Eichhorn M. and Pauly H,. (1975) lnt. J. Radiat. Biol. 28, 401. Schuessler H., Scmerler-Dremel G., Danzer J. and JungKroner E. (1992) Int. J. Radiat. BioL 62, 517. Seki H. and Schnabel W. (1982) Z. Naturforsch. 37e, 63. Shafferman A. and Stein G. (1975) Biochim. Biophys. Acta 416, 287. Simic M. G., Grossman L. and Upton A. C. (1986) Mechanism of DNA Damage and Repair. Plenum Press, New York. Simic M. G., Taylor K. A., Ward J. F. and yon Sonntag C. (1988) Oxygen Radicals in Biology and Medicine. Plenum Press, New York. Solar S. (1985) Radiat. Phys. Chem. 26, 103. von Sonntag C. (1987) The Chemical Basis of Radiation Biology, p. 429. Taylor & Francis, London.

145

Stepan J., Havranek T. and Jojkova K. (1977) Radiat. Res. 70, 406. Symonyan M. A. and Nalbandyan R. M. (1979) Biochem. Biophys. Res. Commun. 90, 1207. Szeinfeld D., De Villiers N. and Wynchank S. (1992) Cancer Biochem. Biophys. 12, 253. Tollin G. and Hazzard J. T. (1991) Arch. Biochem. Biophys.

287, 1. Torchinsky Y. M. (1979) Sulfur in Proteins (Edited by Metzler D.). Pergamon Press, Oxford. Trimble R. B. and Maley F. (1977) J. Biol. Chem. 252, 4409. Verma S. P. and Rastogi A. (1990) Radiat. Res. 122, 130. Viljoen B., Dubery I. A. and Schabort J. C. 0987) lnt. J. Biochem. 19, 837. Wardman P. (1987) In Radiation Chemistry: Principle and Applications (Edited by Farhataziz and Rodgers M. A. J.), p. 565. VCH Publishers, New York. Wardman P. (1989) J. Phys. Chem. Ref. Data 18, 1637. Wary K. K. and Sharan R. N. 0988) J. Radiat. Res. 29, 104. Weinstein M., Alfassi Z. B., De Fellipis M. R., Klapper M. H. and Faraggi M. (1991) Biochim. Biophys. Acta 1076, 73. Whittburn K. and Hoffman M. Z. (1985) Radiat. Phys. Chem. 24, 481. Wilkning V. G., Lal M., Arends M. and Armstrong D. A. (1968) 3". Phys. Chem. 72, 185. Willson R. L. (1982) In Free Radicals, Lipid Peroxidation and Cancer (Edited by McBrien D. C. H. and Slater T. F.), p. 275. Academic Press, London. Winchester R. V. and Lynn K. R. (1970) lnt. J. Radiat. Biol. 17, 541. Winstead J. A. and McNees D. E. (1974) Radiat. Res. 59, 466. Winstead J. A. and Moss S. A. (1972) Radiat. Res. 52, 520. Wolff S. P., Garner A. and Dean R. T. (1986) Trends Biochem. Sci. 11, 27. Yamamoto O. (1992) In Stability of Protein Pharmaceuticals, Part A: Chemical and Physical Pathways of Protein Degradation (Edited by Ahren T. J. and Manning M. C.), Ch. 12. Plenum Press, New York.