Interaction of radiation-generated radicals with myoglobin in aqueous solution—II

Radlat Phrv Chem Vol 23, No 1 2, pp 271 278, 1984 Printed m Great Britain

0146 5724/84 $3 00 + 00 Pergamon Press Ltd

INTERACTION OF RADIATION-GENERATED RADICALS WITH MYOGLOBIN IN AQUEOUS SOLUTION--II ANALYSIS OF PRODUCT YIELDS FOR HYDROXYL RADICALS WITH OXYMYOGLOBIN UNDER DEAERATED CONDITIONS KEVIN D WHITBURNand MORTON Z HOFFMAN Department of Chemistry, Boston Umverslty, Boston, MA 02215, U S.A and IRWIN A. TAUB Food Engmeenng Laboratory, U S Army Natlck Research and Development Laboratories, Natlck, MA 01760, U S A

(Recetved 29 January 1983, accepted 20 April 1983) A~tract--Gamma-lrradmtmn of N20-saturated aqueous solutions contamlng predominantly oxymyoglobm (with small amounts of ferrlmyoglobm and deoxymyoglobm) leads to the near-quanhtatwe conversion of the oxy dertvatwe to the deoxy, fern, and ferrl-peroxlde (ferryl) forms of myoglobln All of the ferrlmyoglobm-peroxlde production is accountable by H202 involvement, ln&catmg OH/H partlopatlon xn the formation of the other product denvatwes A methodology for compositional analysis of the component myoglobln derivatives m this complex system is developed 1 INTRODUCTION COMPARED WITH fernmyoglobln and other haemoproteins, detailed investigation of the interactions of radiation-generated free radicals with oxymyoglobln has been largely overlooked. In an early report, (1) the colour change of red meat samples (dominant in oxymyoglobin) to brown upon exposure to ionizing radiation was described In subsequent studies, irradiation of beef extracts dominant m oxymyoglobin,(2) and of purified oxymyoglobln(34) yielded ferrlmyoglobln as the major product, an attempt at quantification was made by Barton and Johnson. (3) Generation of ferrimyoglobln was shown, furthermore, to be the first step of radiation-induced composRlonal change; at high doses, production of another myoglobin derivative was reported (5) Recently, we have clarified the nature of this secondary product as being a mixture of myoglobln derivatives.(6) At the high doses used in these earlier studies of the radiolysls of oxymyoglobln, mo&fication of the globin and haeme moieties, in sequence, has been observed. (34) Similar results were obtained for irradiation of oxyhaemoglobln solutions. The destruction of the globln moiety in haemoglobin was examined recently as a function of oxygen concentration. (7) Included in this report Js confirmation of radiation-induced conversion of oxyhaemoglobln to the ferri form in aerated solution In addition, conversion of deoxyhaemoglobln to the ferrl derivative was examined quantitatively

Untd very recently, all investigations of radiationreduced changes of oxymyoglobln have been under conditions that are non-specific in free radical identity so that composite reactlvltles of OH and eaq or O~ have been involved. The reaction of e~ with oxymyoglobln has been examined recently by pulse radlolysls(8) where a single-step conversion to ferrlmyoglobln-peroxide has been proposed. As a continuation of our interest m free radical Interactions with myoglobln, we have undertaken a rigorously quantitative investigation of the interact,ons of OH/H with oxymyoglobin, with a focus on mechanistic interpretation. In the present study, we report the nature and yields of the myoglobln derivatives obtained from -OH/H interactions in deaerated solutions dominant in oxymyoglobln. The novel result that substantial formation of deoxymyoglobln occurs is presented. 2. MATERIALS AND METHODS The isolation and purification of bovine muscle oxymyoglobln (MbO2) followed literature procedures (9't°) The purified MbO2 produced only one band upon gel electrophoresls using 7~o polyacrylamlde gel in glyclne-TRIS buffer at pH 8.3 In order to allow spectral-based determination of products in this study, samples of MbO2 were converted to other relevant myoglobln derivatives for spectral characterization In the visible region. Ferrlmyoglobln (Mb m) was produced by oxidizing pure MbO2 with

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Fe(CN)~ and removed by dmlysls and gel filtration chromatography The deoxymyoglobln derivative (Mb H) was generated by addition of excess $20 ] to Mb m Ferrlmyoglobln-peroxlde, l e ferrylmyoglobin (MbW), was produced by addition of a 6 1 molar excess of H202 to the Mb m solution The same spectral product was obtained by addition of a similar excess of H~O2 to the MbO2 solutions at the same pH, the reaction was significantly slower, however Catalase (E C. 1 11 1.6) from bovine hver was obtained as a twice recrystalyzed water suspension from Sigma Chemical C o m p a n y All solutions, buffered with phosphate (0 010 M) at pH 7.3, were made up with water purified by passage through a Mllhpore Purification Tram Solutions were thoroughly deaerated by saturation with N20 at 2 C The solution vessels were 10 mm spectrosll spectrophotometer cuvettes, each with a long graded-seal neck to allow for a hermetic seal with a rubber septum Aeration of irradiated solutions was performed by removing the septum and flushing with air from a Pasteur pipet with rubber bulb The 7-radlolyses were performed at 20 (_+ 2) C using sources with dose rates of 5 5 and 14 7 k r a d m l n ' located at the U S A r m y Natlck Research and Development Laboratories U p o n radlolysls of aqueous solutions, eaq (2 8), O H (2 8), H 2 0 2 (0 7) and H (0 6) are generated The G-values, or molecular yields per 100 eV of energy absorbed, are shown in parentheses To within 4"~, error, the Gvalue may be considered as the # M concentration of a radlolysls product per krad of energy absorbed In N20-saturated solutions, eaq is rapidly converted to OH radical, ~t~ resulting in G ( O H ) = 5 6 Data collection consisted of spectrophotometrlc scans (Cary 118) in the 480-630 mm region before and Immediately after irradiation The timing of each post-irradiation scan was taken as the 545 nm point was traversed Facile numerical computation for compositional analysis, with absorbance data as input. was achieved using a Hewlett Packard 9830A programmable calculator with printer and tape storage capability 3 RESULTS Spectral changes Shown m Fig 1 are the absorption spectra in the wslble region to the red side of the Sorer band of the oxy, deoxy, ferrl and peroxide forms of myoglobin at pH 7 3, the ~-values are based on (ss0(MbO2)= 1 4 6 × 104M 'cm E~9~The fern-peroxide spectrum is that obtained by addition of excess H202 to a M b w solution, the same spectrum is attainable by similar ad&tlon to MbO2 solution The issue of whether this latter product as better described as the peroxide derl-

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vatlve or as a haemlchrome wdl be addressed fully elsewhere (~) Throughout this study, the products of H202 with both M b m and MbO2, which are spectrall> and chemically indistinguishable, ale regarded as the ferri-perox]de (ferryl) derivative ~4) The spectral mformation in Fig. 1 provides the basJs of the an,llytlcal procedure employed in this study U p o n 7-radlolysls of N20-saturated solutions dominant in MbO2, there occurs a depletion of the characteristic M b O 2 peaks m the visible spectrum, accompanied by absorbance growth m the 500, 560 and > 600 nm regions Figure 2 illustrates the observed result for a N20-saturated 28 p M myoglobm solution before and after a dose of 3 7 krad ~s absorbed The initial composition, determined from

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ployed for three components in earlier studies of. OH and e~q interaction with ferrImyoglobln(6)was reliable, extension of the analytical principle to four components based on absorbance measurements at four wavelengths proved unrehable Figure 1 shows the basis of the problem, inasmuch as all four components absorb appreciably in the same wavelength range, while the inherent spectral differences allow analyses of three components to be reliable and consistent, extension to four components loses analytical sensitivity This insensitivity manifests as frequent negative component percentages and as unacceptable total concentration variability ( > 5~o). As a consequence, this type of four-component analysis was not used The analysis of composition developed for this

FiG 3 Spectral changes induced by 3 2krad of y-irradiation in an NzO-saturated 33 pM myoglobm solution Before radiolysls . . . . . , l 6 min after radiolysis (before aeration at 2 5 mm) - - , 3 5-1 09 mm after radiolysis (after aeration) . . . . . Arrows indicate direction of absorbance change absorbance measurements at 490, 560 and 580 nm, is 7 3 ~ M b O 2 , 20~o Mb" and 7 ~ M b ul. After irradiation, the spectrum in the 480-630 n m region was repeatedly scanned for a period of 2 2-16.4 min after the zero-time of radlolysis, taken as the mid-point of the (38sec) radiolysis duration. In this postirradiation period, depletion of absorbance growth in the regions outside this wavelength region is accompanied by absorbance growth m the regions outside this wavelength range. The presence of a significant percentage of M b l[ in the deaerated solutions after irradiation is demonstrated by conversion to M b O 2 following rapid aeration of the solution immediately after the first post-irradiation spectral scan. As shown in Fig. 3, for a 3 3 p M myoglobin solution initially 66~o MbO2, 14~o M b m and 20~o M b n that has been ~,-irradIated with a dose of 3 2 krad, there is, upon aeration, substantial loss of absorbance in the 560 n m region accompanied by growth in the peak regions of M b O 2. A slower post-irradiation process is observed thereafter While this procedure demonstrates the presence of M b ]~ in deaerated solution after irradiation, it is unclear whether any M b H has been produced by the radlolysis of the MbO2 substrate. Further analysis is required to address this issue.

Determmatwn o f M b n composinon In order to convert from the absorbance data to compositional data of the relevant myoglobin derivatives present, the deaerated solutions must be analyzed after irradiation for at least four components, namely MbO2, M b H, M b m and M b TM. Although the Beer's Law analysis of composition that was era-

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system 1s based upon the rapid aeration procedure Illustrated in Fig 3 It consists of two parts, which are combined to give the requisite compositional data In Fig 4(a) is shown the three-component analysis of MbO2. Mb m and Mb TM as a function of time (for the results illustrated in Fig 3) by the Beer's Law method alter aeratton has occurred 2 5 mln after the zero-time of Irradiation This time of aeration, denoted as t.... is taken as the mid-point of the pre- and postaeration spectral scans The composition of each derivative in the aerated solution at t,,~ is then readily determinable by linear extrapolation of the slow composition change with time back to t~,~. As shown in Fig 4(a), the percentages of MbO2, M b ~I~and M b TM at t~,~= 2 5 mln are 48. 37 and 16".~,, respectively In Fig 4(b), the same absorbance data are analyzed for three components. MbO2. M b " and Mb "I, before and after the aeration This procedure clearly omits analysis of Mb ~v However, the percentage of this component IS unchanged by the aeration, as is that of Mb m Thus. the four-component system before aeration and the three-component system after aeration are analyzed for the component derivatives that change composition (MbOe. Mb H) and one of two that does not (Mb m) While the compositional values thereby determined for post-irradiation times on both s~des of the aeration dlscontinmty must be incorrect in an absolute sense, the changes in the component percentages that vary upon aeration (those of Mb H and MbO2) become meaningful In this way. the percentage of Mb ~I undergoing conversion to M b O . is determinable, and hence the percentage composition of Mb" for deaerated solution at t,,~ can be estimated In Fig 4(b), the aeration-induced changes in composition have been determined at t ~ , r = 2 5 m l n , AMb n is 33°,,, A M b O , is 35°0 and AMb m is < Y',, These values are rehable to the extent that the composition of the unanalyzed M b ~v is constant (1 e unchanged by aeration) and that the absolute percentage of Mb ~v IS significantly less than the other components Over the studied dose range, the selfconsistency of the AMb H and AMbO2 values (usually within 10')o), and the observed constancy of the Mb IH composition (within 5°0) through the aeration &scontinuity, together sustain c o n f d e n c e in the analytical method The average of two A-values is taken as the composition of M b " in deaerated solution at tdlr As a further check on the efficacy of this analytical procedure, the same absorbance data (shown m Fig 3) were analyzed by the Beer's Law method for MbO2, Mb" and Mb ~v before and after the aeration In this case, the two changing components are again analyzed along with a third component that is lnsen-

Sltlve to the aeration The change m composition associated with the M b " - + M b O . conversion agreed within 10"o error with the previous method for concentratlon-normahzed doses up to 0 0 5 k r a d I*M r Beyond this dose range, this second procedure Introduced negative compositions m order to accomodate the diminished absorbance, especially after aeration, in the 550 600 nm regmn accompanying a significant Mb m build-up This inconsistency is a consequence of the fact that MbO:. and Mb m and Mb ~v all absorb appreciably in a region where Mb "~. the unanalyzed component, does not (see Fig 1) Consequently, for greater consistency and rehablhty. the Mb I~-+MbO~ compositional {hange accompanying aeratmn is based on the analysis of MbO> Mb" and Mb m m this investigation Having determined the percentage composition ot Mb H for deaerated solution at t,,~ by the aeration procedure, subtraction of the initial percentage of Mb ~fprior to irradiation allows the percentage of Mb generated (or lost) by the radlolysls to be estimated for the same conditions The MbO2 composition determined in Fig 4(a) for aerated solution at t.,~ can then be corrected for the M b " - + M b O . conversmn reduced by the aeration Specifically, subtraction of the Mb ~ composition at t,,, determined In Fig 4(b) gives a value of the MbO~ composition tbr deaerated solution at t,,~ As a result of the analytical procedure described thus far. the compositions of MbO2, Mb". Mb m and Mb Iv are determined to be 13. 34. 37 and 16°,,. respectively, for deaerated solution at t~,~- 2 5 rain The difference between the solution composition before irradiation and at t,,~ can be determined next to be a 5Y',, loss of M b O > and increases of Mb m, Mb I~ and Mb fv of 23, 14 and 16°,. respectively Radtolyst.s vtel& The difference In composition determined at t~,r relative to the initial composition before Irradiation is a result of two factors First, there IS radiationinduced compositional change, secondly, there is ,i subsequent post-irradiation change Extrapolation of the compositional data determined at several values of td,r back to the zero-time of radlolysls provides a means of removing the post-irradiation effects Thereby the changes induced by irradiation can be estimated, these changes are fundamental to any mechanistic interpretation of MbO2 radlolysls This approach is Illustrated in Fig 5 for the concentrationnormalized dose of 010 k r a d t t M ~, the compositional changes determined from Fig 4 at td.r = 2 5 are shown here, along with those for separate experiments having t,,r values of 3 9 and 5 7 m l n s Compositional chan~es at the "zero" time of radmlv~t~ ~1"

Product y~elds for hydroxyl radicals with oxymyoglobm

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MbO2, Mb "t, Mb H and Mb w of 53, 17, 22 and 15~o, respectively, are indicated for this dose These values are estimates of the radtation-mduced changes m composition for the four relevant components As illustrated m Fig. 5, the dominant post-irradiation process occurring among the myoglobin derivatives in deaerated solution appears to be MbH-,Mb H~ conversion Whether this observation, which is a general one over the studied dose range, corresponds to a conversion directly involving modified Mb, indirectly mvolwng unreacted H202, or represents the composite of complex cross-reacnons among myoglobm denvatwes cannot be determined from the bruited results A dose profile of the radmuon-mduced changes m composition for solutions mitmlly averaging 65% MbO2,26% Mb Hand 12~oMb l " , l s s h o w n m Fig 6 Using the relationship that G(product)-~ #M (product) per krad, determination of the slopes of the linear plots of ~-composltlon vs concentrationnormahzed dose allows the G-values of production of Mb u, Mb I" and Mb Iv and of the loss of MbO2 to be determined The G-values determined by leastsquares analysis are, respectively, 1 9 ( + 0 3), 15 ( + 0 2), 0 8 (+_0 2), and - 4 . 2 ( + 0 3) for the speofied experimental condmons

Effect of catalase on product ywlds The observed co-production of Mb zv may m prmople derive from the interaction of H202 with MbO2 and/or Mb m, or from a direct interaction of OH/H

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FIG 6 G-value determination from plots of')o-composltlon change vs concentratlon-normahzed dose (a) oxymyoglobm, O, deoxymyoglobm, A, (b) fernmyoglobm, ©, fernmyoglobm-peromde, • with MbO2 In order to resolve this issue, N20-saturated solutions dominant in MbO 2 were irradiated in the presence of bovine liver catalase at a concentration of ~ 20 pg ml ~. This enzyme rapidly dlsmutates H202 The [catalase] was chosen to satisfy three important criteria, that, at this concentranon, < 4'30 of the available OH/H radicals react with catalase in compennon with myoglobln, assuming k( OH/H + S) is similar for both substrates, that > 90% of the H202 reacts with the enzyme rather than with myoglobin at th~s concentration; and that catalase absorbs < 5°~, of the myoglobln absorbance m the analyzed spectral region at the chosen concentraUon The catalase-contammg solunons of 29 (+_ 2) p M myoglobin were irradiated and analyzed as before by the determmanon of the composmon of deaerated solutions at the Ume of aeranon. Greatly d~mmlshed post-~rradmUon reacnwty among the myoglobin derivatives is observed This feature ehmmates the need to extrapolate this reacnvlty over several data sets at d~fferent t..r values. Consequently, the solunon composmon determined at the shortest value of t..r 1S taken as that at the zero-Ume of radlolys~s, ~ e the radmUon-mduced change, this approximation lntro-

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duces lnstgnificant error. N20-saturated solutions initially averaging 6 4 ~ M b O 2 , 24~oMb 1~ and 12~o M b m were analyzed by the Beer's Law method over a dose range of 0.62-3.3 krad, the dose profiles are shown In Fig. 7. A least-squares analysis of the radiation-induced changes in the composition as a function of concentration-normalized dose gives G(-MbO2)=-3 3 ( + 0 2 ) , G ( M b ' ) = 1.4 (+0.2), G ( M b " I ) = 1 7 (-I-02) and G(MbXV)<0.1 for the experimental conditions. The virtual elimination of M b ~v production by the presence of catalase suggests that this product IS derived from H202 Involvement, and that production via a pathway involving the free radical interaction is insignificant Smce the irradiated system slmphfies to a three-

TABLE

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G-VALUES

FOR CHANGE

component solution in the presence of catalase, these same absorbance results were analyzed directly for the MbO2, Mb H and M b H~ derlvatwes before aeration. Dose profiles of the composmonal data obtained in this way are shown m F]g 8, from which the following G-values are obtained by least-squares analys]s. G ( - M b O 2 ) = - 3 4 ( + 0 2), G ( M b H) = 1 7 ( + 0 2 ) , and G ( M b N I ) = I . 7 (-+02) These values agree well with those obtained by the indirect (aeration) analysis, and thereby lend further support to the efficacy of the methodology used m the absence of catalase Results s u m m a r y

The radlolysls yields determined in the absence and presence of catalase are summarized in Table 1 4 DISCUSSION The G ( - M b O 2 ) - ~ - 4 2 observed m the absence of catalase is for a system initially ~63~o m MbO2 and ~ 37~ in the M b TM and Mb H deravatwes Assuming similar rate constants for O H / H + myo-

I N C O M P O S I T I O N IN " ~ - I R R A D I A T E D N 2 0 - S A T U R A T E D

MYOGLOBIN SOLUTIONS

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Analytical Method

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Product yields for hydroxyl radicals with oxymyoglobin globln regardless of the nature of the derivative, then only ~ 63~o of the available. OH/H. radicals actually react with the MbO2 substrate while the remainder attack the other derivatives present initially. Inherent in the assumption is that the free radical attack is predominantly at globln moieties as discussed m the case of the OH/H- reaction with ferrlmyoglobin.(6) Correction of the observed G-value to 100~o OH/H interaction with MbO2 gives a value of G(-MbO2)--6.9, which is the sum of G ( O H ) + G(.H) + G(H202) in the N20-saturated system. This result is consistent with quantitative involvement of .OH/H' and H202 in the overall observed loss of MbO2 induced by y-irradiation In the absence of catalase. The analogous correction of G(-MbO2)=-3.3 for an initial solution compositron ~ 64~o in MbO2 in the presence of catalase to 100~o free radical attack on MbO2 gaves a G-value of ~ - 5 2 In this case, near quantitative conversion of MbO2 to other myoglobln derivatives is indicated. Inasmuch as Mb ~ and Mb I" are present inltmlly at minor concentrations, the observed G-values include a contribution to change in composition from Mb"~_Mb"l~-Mb w lnterconverslons upon y-lrradmtlon,16) an addition to changes deriving from MbO2 The previously determined efficiencles of the M b " ~ M b m and Mbm--*Mb" conversions Induced by OH/H are rh = 0.55 and t/_~ =0.23, respectively. Using these efficiency values and the initial composition data (26~o Mb", 11~ Mbln), the G-value for the compositmnal change denving from these minor components, expressed as G'(Mb"I), can be calculated using expression I (1) where

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Noting G"( OH/H ") is 6.2 in N20-saturated solution, the calculatmn gives G'(Mb m) "" 0.27 for an overall M b " ~ M b m conversion between the minor components Comparison of this calculated value with the observed G-values obtained in this study indicates that the observed G(Mb") is low and the observed G(Mb m) is correspondingly high relative to the situation that would be attained in the absence of initial concentrations of Mb" and Mb m in solution. Consequently the source of Mb" in these y-irradiated solutions is MbO2 through .OH/H' interaction. Moreover, since G'(Mb "I) is significantly less than the observed G(Mbm), substantial conversion of M b O 2 ~ M b I" is also indicated by the results.

277

The mechanisms of product formation may be considered in terms of initial OH/H attack predominantly on the globln moiety of MbO2 A range of abstraction and adduct radicals on the protein residues would result as a consequence Those secondary radicals in favorable conformation with the heme having sufficient reducing potential can reduce the bound O2 of MbO2 in a first step, along with concommltant oxidation of the protein residue Subsequent adjacent electron-transfer from the Fe" center to the reduced bound O2 would complete a two-equivalent reduction of this oxygen and generate a Fe In heme in the process. These steps are shown in reactions (1)-(3), where MbO2 is represented as PFenO2 and P designates hemeprotein. (1)

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The analogous composite of reactions (2) and (3) has been previously proposed in the oxidation of oxyhemes by reducing agents (15) In addition to this conversion to globin-modified ferrl-Mb, the secondary globin radicals may follow another course of reactivity Specifically, this involves self-scavenging by the labile bound O2 molecule (4)

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Rates constants for reactions of 02 with abstraction and adduct radicals of ~ 1 0 9 M -I sec i are known (16) The production of peroxy intermediates probably terminates heme-centered redox because of a marked unfavorable change in reduction potential of the radical rate The likely fate of these radtcals is bimolecular decay to generate oxidative modification of the globm moiety. The rigorously quantitative results obtained in this study demonstrate that both ferrlmyoglobln and deoxymyoglobln are the major products of the interaction of ra&atlon-generated -OH/H' radicals with deaerated solutions predominant In oxymyoglobln Co-production of ferrimyoglobin-peroxlde (ferrylmyoglobm) apparently derives from the H202 that is generated during irradiation. Quantitative conversion of oxymyoglobin to these product derivatives ts indicated. Determination of the intnnsic efficxencies of the MbO2--*Mb~l and M b O 2 ~ M b In conversions induced by OH/H. and details of the likely mechanisms of product formation will be reported elsewhere

278

K D WHITBURNel al

A~knowledgement~s--Asslstance In the preparation of the purified myoglobln extracts by Dr J J Shleh is gratefully acknowledged This research was supported m part by U S Army contract D A A G 29-82-K-0132 to Boston University

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