Free Radical Reactions with Simple Biochemical Systems J. Leslie Redpath Department of Radiological Sciences, University of California, Irvine. Irvine. CA 92715
Free radical processes are an integral part of biochemical processes and as such play a role in the development of a variety of biological effects and pathological disorders. For example, free radical mediated lipid peroxidation has been implicated in cellular aging processes, liver damage induced by chlorinated hydrocarbons and alcohol, cellular damage hy air pollutants and oxygen toxicity (1 and references therein). In addition, free radical processes are known to be involved in radiation-induced cellular lethality (2 and references therein) and most probably involved in the initiation of the carcinogenic and mutagenic action of ionizing radiation. The carcinogenic effect of certain polycyclic hydrocarbons is also thought to be mediated through free radical processes. For examole. . . certain chemical carcinoeens are converted to active forms by enzymalir pnressei i n w l v i t ~ t :free radical reactions (3).This sameactivnlim ~mwt.~si.;an l w achieved bv exuwinr the biological system and "inactive" carcinogen to ior~izing radiation ( 4 ) . T h e above paragraph outlines some biological effects involving free radical processes. Most of the effects mentioned are undesirable. By understanding the mechanism of free radical reactions with biochemical systems, means of controlling these reactions should he able to he devised, and thus be of benefit to mankind. An example of this is in the radiation therapy of cancer where radiation sensitizing and protective agents are being used in attempts to increase tumor cell kill while preserving acceptable levels of normal tissue injury ( 5 ) . These sensitizing and protective agents act a t the free radical level. Ionizing Radiation-A Convenient Means of Generating Free Radicals As we have seen in the preceding papers in this symposium ionizing radiation provides a convenient way of generating free radicals. Therefore radiation chemical methods can be used readily to study free radical reactions in whatever chemical system is of interest to you. I t is important to realize here that
ionizing radiation can be used as a "tool" to study free radical reactions. Radiation Chemical Methods in the Study of Free Radical Reactions In order to avoid repetition of what has been described in the preceding papers I will only briefly survey this topic in this section. Since most hiochemical systems we deal with are aaueous solutions. the radiation chemistrv of water becomes the resulting ions undergo a variety of ion-molecule reactions to produce what are known as the primary water radicals, namely OH', the hydroxyl radical; e-.,, the solvated electron, and H', the hydrogen atom. HzO
-
H20+
H10 + e-+.OH,
e-.,, -H
(1)
These soecies are themselves verv reactive. and therefore short-lived, and in the ahsence of added solute will react with each other to form the permanent products of water radiolysis, hydrogen (Hz), and hydrogen peroxide (Hz02). Immediatelv follow in^ an absorbed dose of radiation of 1000 rad there will be -3 p ~ b 'OH f and e-, radicals produced and 4 . 5 pM .H atoms. The hydroxyl radical is a strong oxidizing agent while the hydrated electron is a strong reducing agent. The hydrogen atom can react either as a reducing agent or an oxidizing agent. Fortunately, there exist several solutes which will react rapidly with 'OH and slowly with ec,, and vice versa. Thus by adding an appropriate concentration of such a solute to the system under study it is possible to study the reactions of one radical independently of the olher. Scavenger systems which allow us to do this are listed in Table 1. Of course the reaction of a ~ r i m a r vradical with a solute will produce a solute radical which may or may not be reactive with the system under study. For example, both tertiary butyl alcohol and bromide ions are excellent .OH scavengers; however, the secondary radicals produced by OH reaction, namely
Volume 58
Number 2
February 1981
131
'CH?rCH;&H mrl Hr. , (Iliier uidely In their recpl.t I I W reactivirie.;. 'l'he tt,rtl;jry l ~ u t R~I Cl O ~ O I radical is r ~ l i ~ t i w I \ unreactive, wherr:15 tlw l l r , radical anwn is a itrml: crxi dizing agent. Free Radical Reactions with Amino Acids, Peptides, and Enzymes The object of this paper is to discuss free radical reactions in biochemical systems as studied using radiation chemical techniques. T o attain this end I have chosen not to give an exhaustive survey of this subject but rather to illustrate it principally with specific examples from my own work (in collaboration with several colleagues) on free radical reactions with enzyme systems. Before doing this, however, I will briefly describe the nature and function of enzymes in biochemistrv. Enzymes arc lnrgt- prdcin nidecules essentially made up o i a lone chain d , i m i w : i d s i n a weciilc cunfiyurafi(m. '1'ht.y control specific chemical reactions by forming a complex with a substrate. allowina chemical reactions to occur with the liberation of aprod&t together with enzyme. In this sense, the enzyme is a catalyst. There is gentmlly 81 slwcilic iitr wirhin an enzyme mnler~~lr. to w h i c h a s u h s t r n t e ~ mh n d : ~ n dat which thechemisrrywcurs. This site eenerallv involves several amino acid residues lying in a specific configuration with respect to each other. These residues although physically located close to each other may he separated by many other residues in terms of their oosition in the . oolvoe~tide .. . chain. The experiments I will describe in the paragraphs below illustrate how ionizing radiation can be used as a tool to probe for the nature of the amino acid residues involved in the chemical and structural function of enzvmes. It is appropriate to start with an ill&ration that water derived free radicals are effective enzyme inactivators. This is readily demonstrated by comparing the radiation sensitivity of dry enzymes with those in dilute aqueous solution. Figure 1compares the radiation inactivation of dry rihnnuclease with the same enzyme in dilute aqueous solution. The increased sensitivity in solution relative to the dry state can be explained by the reaction of water free radicals, particularly 'OH and .H radicals. Reactions of Primary Water Radicals with Amino Acids, Peptides and Enzymes During the past 10 to 15 years a great deal of information has been gathered regarding the kinetics and mechanism of reaction of the primary water radicals with amino acids and ~
~
~
~
~~
Reactions of Selective Secondary Radicals with Amino Acids, Peptides and Enzymes The use of selective free radicals to identifv amino acid specific functional groups. Such selective free radicals were first recognized about 10 years ago through an unexpected result in an experiment designed to demonstrate the protection of lysozyme, irradiated in an NzO-saturated solution, against 'OH radical inactivation by the OH scavenger CNS(9).The rate of enzyme inactivation (Fig. 2) was unaffected although all 'OH radicals had been converted to the radical anion .(CNS)z- by reactions (3) and (4), viz.:
-
.OH+ CNS- OH- + CNS (3) 'CNS + C N S * (CNS)?(4) The only explanation of this result was that the '(CNS)z-
60-
RIBONUCLEASE
40-
20-
iqxides. This subject ha; hren rk\ wn~:dI,? ceveral authms and readers are relcrred t o t l ~ r i evntx.rr ior detailc. There is a wide spectrum of reactiv&s encountered for each of the radicals with the 20 commonly occurring amino acids. The aromatic and sulfur-containing amino acids tend to he the most reactive, often reacting with diffusion controlled rates. The rate constants are pH dependent due to the various ionic equilibria in amino acid structures. Hydrated electrons will react with ~ r o t o n a t e damino erouos and a t carbonyl groups in peptide;. Hydroxyl radicalsand'hydrogen atoms will often react by H-atom abstraction from carbon atoms alpha to the carhoxyl group (in free amino acids) or to the peptide bond (in peptides). The simple aliphatic compounds glycine and alanine are the least reactive; however, when OH groups or branched chains are present the reactivity tends to be increased due to inductive effects. Since an enzyme consists of many different amino acid residues.. . orimarv water radicals will react a t manv different sites on the enzyme hecause of the general lack of specificity of reaction of these radicals with amino acid moieties especially when in peptide form (6, 7). While by the use of appropriate scavenger systems (see Table 1)it is possible t o assess the relative contrihutions of the radicals ('OH, e-., .H) to enzyme inactivation, it is not pnssihle to state a t which amino acid residues critical damage occurred ( 8 ) . Free radical inactivation of enzymes can occur by several possible mechanisms. Firstly, damage can occur to an amino acid residue or residues in the active site which is either involved in substrate binding or in the chemistry of the catalytic reaction. Secondly, damage can occur to residues which are e y n ~ i ~ . ~ c r i rHy i r y[he . useof r;lrlid!z~call\~ ...eenerated freer;~dic:,l\whirh nrc snecific in their react~onswith amino acids, it is possible to determine residues in an enzvme which are critical to enzvme function. T h e experimental approach in such an instance is to combine information from oulse radiolvsis exoeriments on rates of rei l c i ~ mand sites ofattack with stuti31~1rv-swte radiolysisenwme inactivation data. In thewcti#mhelow I will describe the use of selective secondary radicals in probing enzyme structure and function. th.
Table 1. Some Scavenger Systems and Resulting Reactive Radicals
SOLUTION Srnglrnl
Scavenger System
Reactive Radicalss
N2 + t-butyl alcohol (0.1M) N@ + t-butyl alcohol (0.1M) N@ + CNS- (0.1 M) N 2 0 + Err (0.1 M) N@ + HCOO- (0.1 M)
'OH (2.7).e y q (2.7).'H (0.55) 'OH (5.4). 'H (0.55) e y , (2.7). H (0.55) H (0.55) '(CNS)2(5.4). 'H (0.55) B r , (5.4). H (0.55) COz- (5.41,H (0.551
N2
N20
10-
2
4
6
8
10
12
DOSE Mrad Figure 1, lnaetivation of ribonuclease in the dry state and in aqueous solution by %o y-radiation. (Redrawn horn Dertingar 8 Jung ( 16).) 132
Journal of Chemical Education
Numbers in parentheses are Gvalve~ (radicals1i00ev).
are pau!elqo sqnsar pue paleB!lsanu! S & ~ S K Saql JO amos PUB 'arnqeial!1 aql u! paq!msap uaaq aneq sawKzua [eraAas roj
jo uo!pear Bu!molloj sqanpord aqq jo eiqsads lua!suerl aql Bu!redruoa pue Bu!rnseam Kq paleqsuowap sem smaao paapu! s!ql 1eql p e j aq& .amkzoskl u! sanppai u e q d q -d&q az!p!xo plnom -7SN3). qeql LTdLLI! plnom Sp!aB OU!WB 1enp!~!pu! q l ! ~q e p a!lau!~ aq& '~ea!per Krepuoaas an!?aalas n S! -Z(SN~).snq& .sp!ae ou!me raqqo kue qqm ueql ralsej satuq 001 0%09 wer e qql!fi oeqdoldLl3 q l ! ~palasar -Z(SN~). ~d [erlnau $8p q l luaredde aweaaq I! anb!uqaal s!sK[o!pei aqnd aqq Bu!sn sp!ae o u y e ql!m -Z(SN~).JOi(l!n!qaeai aq7 JO Karuns e uodn .awKzua aqlZu!len!laeu! aq lsnw uo!ue p p e i
YOI*F!UIB~ 411" 101 I U0.l ln3nmKIW) EpB100t.Z '.Ma ' s l n d aql l a w 3aSd os w m e a u mmls M . otl suonbe a s w o s a , a w p a w c . o ~~~w d o t d b ~ O S J Oos ! ~W I ~ ~ P S - O < 10 N54h,o*p~>as1nd u o l l 9115eds lualsuell P am614
Hl3Nm3AVM 0?_E7 Oy.-O$S
WU
009
005
00V
OOV
OOE,
(CNS)? + PAPAIN pH 1 1 1
I
, 300
I 400
500
600
WAVELENGTH nm Figwe 6. Transient spectra from pulse radiolysis of NIOaaturated solutions of papain (0.75-1.0 mg/m~)containing KCNS (5 X l o + M). Spectra measured 50 &saner a 1000 rads pulse.
20
L0 60 80 NUMBER OF PULSES Figure 8. Elachon transfer Com the formate radical C02-, to sulphur bridges in ribonuclease. Main figure: Effect of number of pulses on the absorption of RSSR- at 410 nm. Ribonuclease concentration 2.0 mglml. formate concentration = 4 X M. pH 8.0. inset: Oscillogram showing rate of formation of RSSR- at 410 nm. Each curve recorded aner indicated number of prepulses. Dose per pulse = 2 Krad. Sweep speed 20 &seconds/cm. (Reproduced from ( 8 ) with perrnisrsion from the Israel J. Chemistry and the Weizmann Science Press at Israel).
Table 2. Reactlvlty of Some Amino Acids with Radical-Anions Radical Anion
Tryptophan
Tyrosine
Histidine
6-Aianine
Cystelne
Melhionine
Figure 7. Shuctural inhibition of elecbon transfer fmm the formate radical im to the S-S bridges in enzymes (see text).
*
Reaction of Selective Reducing Radicals with Amino Acids and Enzymes The hydrated electron is a reducing radical which reacts rapidly with many amino acids, in particular histidine and oxidized cysteine, i.e., cystine. The formate radical ion, Con-, formed by reaction of hydroxyl radicals with formate, viz.,
-
.OH + HCOO- 'CO1- + H20 (5) is a more selective reducing species than the hydrated electron. At neutral pH this radical reacts with disulfide bridges with a rate constant of -lo9 M-' sec-I and with all other amino acids a t
