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Radiation induced modification of tryptophan and tyrosine residues in flavocytochrome b2 in dilute aqueous solution
Radiation Physics and Chemistry 59 (2000) 71±80
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Radiation induced modi®cation of tryptophan and tyrosine residues in ¯avocytochrome b2 in dilute aqueous solution D. Bhattacharya, A. Saha, P.C. Mandal* Nuclear Chemistry Division, Saha Institute of Nuclear Physics, 1/AF Bidhan nagar, Calcutta, 700 064, India Received 12 August 1999; accepted 10 November 1999
Abstract Steady state gamma irradiation of an aqueous solution of ¯avocytochrome b2 under dierent conditions led to modi®cation of tryptophan and tyrosine residues. These aromatic amino acid residues were more susceptible to the attack by OH radicals than H atoms. Unchanged quantum yield values for tryptophan and tyrosine residues and unchanged tryptophan excited state lifetime in the irradiated enzyme suggests that irradiation results in breakage of some non-covalent bonds disrupting the peptide framework partially. It is justi®ed by the circular dichroic studies for the irradiated enzyme which shows a reduced helicity but no evolution towards any other structures. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Flavocytochrome b2; Tryptophan; Tyrosine; Fluorescence quenching
1. Introduction Flavocytochrome b2, an integral member of the alternative respiratory chain in yeast (Pajot and Claisse, 1974) has been the centre of a ¯ourishing area of research (Miles et al., 1992; Silvestrini et al., 1993; White et al., 1993; Rouviere-Fourmy et al., 1994; Sharp et al., 1994). Characterization of its crystal structure (Xia and Mathews, 1990) at 0.24 nm resolution made it possible in unraveling of its catalytic, bio-physical and molecular biological aspects in ®ner details (Chapman et al., 1991; Balme and Lederer, 1994). However the eects of ionizing radiation on these systems remain an open question. Flavocytochrome b2, ideally belongs to the group of
* Corresponding author. Tel. +91-033-3370605; fax: +091033-3374637. E-mail address: [email protected] (P.C. Mandal).
lactate dehydrogenases (LDH) with wide digressions in structural aspects. Radiation-induced primary water radicals are known to attack at dierent sites in the enzyme. LDH, in fact, were found to undergo residue modi®cations and conformational changes which was believed to trigger due to radiation induced peptide chain breakage (Winstead and Reece, 1970; Schuessler and Denkl, 1972; Schuessler et al., 1975; Buchanan and Armstrong, 1976). It would be reasonable to anticipate that a signi®cant contribution of some speci®c reactions of this type may result in residue destruction or modi®cation which in turn might lead to changes in the enzyme secondary structure. The current work aims at elucidating the same in the ¯avin and hemelinked LDH, ¯avocytochrome b2 and relate it to the radiolytic responses observed in other LDH molecules. Modi®cations of tryptophan and tyrosine residues in enzymes irradiated with g-rays under dierent conditions were measured by change in ¯uorescence of tryptophan and tyrosine and also from second deriva-
0969-806X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 9 9 ) 0 0 5 2 0 - 4
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
tive absorption measurements. Any change in the conformations of the enzyme after irradiation was ascertained by CD studies.
2. Materials and Methods 2.1. Chemical Flavocytochrome b2 (from Saccharomyces cerevisiae ) E.C. 1.1.2.3, FMN, N-acetyl tyrosinamide and guanidinium chloride (GdmCl) were purchased from Sigma Chemical Co., USA. Phosphates and b-mercapto ethanol (2 ME) were of analar grade. 2.2. Desalting of the enzyme solution The required amount of enzyme suspension in 3.2 mol dmÿ3 ammonium sulphate solution was drawn out carefully and centrifuged. The precipitate thus obtained was dissolved in neutral 0.1 mol dmÿ3 phosphate buer after washing it twice in 70% ammonium sulphate (Lederer, 1974) was subsequently irradiated.
which otherwise tends to shift due to interfering contributions from Trp at this wavelength, thus making the analysis virtually impossible. The contributions of added Tyr in the form of N-acetyl tyrosinamide were taken into account during the calculation. The measurement was made using a Shimadzu 2101 spectrophotometer, the automatic correction mode was used routinely and the other settings are: slit set at 0.2 nm and the scan speed slow. 2.6. Quantum yields The quantum yields f, for Trp and Tyr were obtained by excitation at 280 nm or 297 nm, by using the relation, AE =AR fE ODE n2R =fR ODR n2E where A is the area under the curve, OD is the optical density, n is the refractive index, subscripts E and R refer to enzyme and reference substances respectively, free Trp and free Tyr were used as reference in neutral, phosphate buer, f values for both being 0.14 (Cantor and Schimmel, 1980; Pinho Melo et al., 1996).
2.3. Irradiation Procedure A 60Co g-source with an absorbed dose rate of 5.5 Gy/min determined by Fricke dosimeter (Spinks and Woods, 1976) was used, for irradiating the enzyme solution. 2.4. Steady state ¯uorescence measurements The ¯uorescence emission spectra were recorded with a Hitachi spectro¯uorimeter with entrance and exit slits set at 5 nm. The ¯uorescence emission intensity of tryptophan (Trp) only for excitation at 280 nm was obtained by normalization of the spectrum of lexc (297 nm) at 380 nm relative to that of lexc (280 nm) at 380 nm. Subsequent subtraction of the former from the latter gave the ¯uorescence emission due to tyrosine (Tyr) only (Pinho Melo et al., 1996). 2.5. Second derivative absorption studies The second derivative absorbance at a particular wavelength is the algebraic sum of the contributions of dierent components at that wavelength only. The technique employed here aims at determining the vertical distance from the baseline to the second derivative curve (Nozaki, 1990) at a wavelength assigned to a particular amino acid (283 nm for Tyr). N-acetyl tyrosinamide was used as a titrant that helps to retain the constancy of the trough position at 283 nm due to Tyr
2.7. Lifetime measurements The single photon counting technique was used to study the total intensity decays. The instrument was set up in our laboratory using the components supplied by Edinburgh Instruments (model 199F time domain ¯uorimeter). The decay pro®les of Trp in native, irradiated and denatured enzymes were obtained for 2500 photon counts at lem (335 nm) for lexc (297 nm). Lifetimes were estimated by ®tting each individual pro®le to the sum of two exponential decays and the accepted values correspond to the minimum w 2. Mean lifetimes were calculated by hti=a1t1+a2t2 where a is the fractional amplitude of the decay component with lifetime t. 2.8. Circular dichroic studies CD spectra for native and irradiated enzyme solutions were collected using a Jasco J-700 spectropolarimeter in quartz cells of 1 mm path length in the far UV range (200±250 nm). Five measurements were made for each sample at a scan speed of 20 nm/min, 2 s response time and step resolution of 0.1 nm. The spectra were corrected for the baseline by subtraction of the contribution due to blank solution (buer free from enzyme). The data were expressed in terms of mean residue ellipticity as deg cm2 dmolÿ1.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
3. Results and discussion Akin to biological systems, radiation induced damages of ¯avocytochrome b2 were studied in a dilute aqueous environment (0.1 mol dmÿ3 phosphate buer, pH 7.0). The high energy radiation is absorbed by the solvent, in this case, with the production of a number of primary water-derived radicals, namely hydrated electrons (eÿ aq), H atoms and OH radicals. In the absence of oxygen, each individual species has a characteristic eciency of its own such that the eciency of ¯uorophore loss can be designated as (Lakowicz, 1983) G -Fluorophore Sfi gi
1
where fi is the fractional eciency of the residue modi®cation and G1 (-Fluorophore) indicates the yield of the ¯uorophore loss under the same irradiation condition while gi is the primary yield of the respective radicals. In argon and N2O saturated conditions, according to Eq. (1) G(-Fluorophore) would be due to contribution from all the three radicals derived upon g-irradiation of an aqueous solution such that, G -Fluorophore fOH gOH fH gH feÿaq geÿaq
2
In N2O saturated medium, however, eÿ aq is converted to an equivalent amount of OH radicals, ÿ eÿ aq N2 O 4 OH OH N2
k 0:91 1010
dm3 molÿ1 sÿ1 Buxton et al:, 1988
3
such that Eq. (2) boils down to G(-Fluorophore)=fOHgOH '+fHgH gOH ' being the total yield of OH radicals indicating that there is an abundance of OH radicals in an N2O saturated medium. Since the studies are carried out in 0.1 mol dmÿ3 phosphate buer, hence in an argon saturated medium ÿ 2ÿ eÿ aq H2 PO4 4 H HPO4
k 1:9 107 dm3
molÿ1 sÿ1 Ye and Schuler, 1986
4
such that there is a predominance of H atoms, G -Fluorophore fOH gOH fH gH 0
5
where gH ' is the sum total of gH and geÿaq. 3.1. Fluorimetric studies To assess and quantitate the extent of damage of
1
G-unit=0.1034 mmol/J.
73
Trp and the Tyr residues after irradiation of ¯avocytochrome b2 ¯uorescence emission of the enzyme was followed before and after irradiation in 0.1 mol dmÿ3 phosphate buer with lex(297 nm) and lex (280 nm) (Fig. 1). The lmax observed at 335 nm (Fig. 1A(a)) is entirely due to the Trp residues in the enzyme which is a characteristic feature of a protein with a large number of Trp residues, the blue shift from that of free Trp (lmax 350 nm) is due to the shielding of the Trp residues from water molecules by the protein matrix. Interestingly irradiating the enzyme up to a dose of 12 or 18 Gy (Fig. 1A and 1C respectively) does not indicate any change in the ¯uorescence emission spectrum in a N2O saturated medium, though a considerable reduction in the emission intensity occurs. An almost unchanged lmax for an irradiated enzyme indicates that practically no enhancement of polarity occurs. When the ¯uorescence emission of native enzyme was followed, after denaturing it in 6 mol dmÿ3 GdmCl and 2 ME and exciting at 280 nm lmax for emission registers a red shift (350 nm) with the appearance of another distinct peak at 310 nm due to the Tyr moieties in the enzyme (Fig. 1A(d)). Now, when the concentration of both the irradiated and the unirradiated samples are the same, it is possible to judge the extent of their damage from the ratio of the ¯uorescence intensities of the unirradiated sample to the irradiated one (Saha et al., 1993) both denatured in 6 mol dmÿ3 GdmCl and 2 ME. The ¯uorescence intensity of both Trp (Fig. 2A) and Tyr (Fig. 2B) residues in the irradiated samples (in 0.1 mol dmÿ3 phosphate buer, pH 7.0) under dierent conditions in the presence of the denaturant decreased exponentially with increasing dose indicating linear semi-log plots in Fig. 2. When irradiation is carried out in aerated medium, molecular oxygen converts H atoms and eÿ aq to HO2 ÿ and O2 radicals respectively (Buxton et al., 1988). Literature values (Buxton, 1987; Bielski et al., 1985) show these two radicals do not react with Trp and Tyr. Therefore OH radicals alone are responsible for
Table 1 Number of Trp residues in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 after irradiating at D50 for each of the three conditions (aerated: 55 Gy, argon: 11 Gy and N2O: 28 Gy)a Irradiation conditions
Number of Trp residues
Native Aerated Nitrous oxide saturated Argon saturated
5 4 3 4
a Experimental uncertainty: 210%. All results are an average of at least three sets of experiments.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Fig. 1. (A). Corrected and normalized spectra of 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in 0.1 mol dmÿ3 phosphate buer (pH 7.0). The four spectra depicted here represents both native and denatured enzymes. Spectra (a) and (b) represents that of native enzyme with an excitation at (a) 297 nm and (b) 280 nm. (c) and (d) represents the ¯uorescence emission spectrum of enzyme denatured in the presence of 6 mol dmÿ3 GdmCl and 2 ME with an excitation of (c) 297 nm and (d) 280 nm, (B) Fluorescence emission spectra (excitation at 297 nm) of ¯avocytochrome b2 in 0.1 mol dmÿ3 phosphate buer (pH 7.0) irradiated under N2O saturated condition at dierent doses in the absence of the denaturant, (C) same as in (B) but after denaturing in 6 mol dmÿ3 GdmCl in the presence of 2 ME.
the damage in this case. Rate constants of Trp at neutral pH with OH is 1.3 1010 dm3 molÿ1 sÿ1 (Solar et al.,1984a; Geto, 1992) whereas that with H atoms is 2 109 dm3 molÿ1 sÿ1 (Kirby and Steiner, 1970) explains the greater extent of residue damage in N2O saturated conditions (Table 1) where there is an abundance of OH radicals. Exposure to a very high dose (D502, 55 Gy) (Bhattacharya et al., 1995) in aerated medium possibly allows a greater interaction of the OH radicals with the Trp residues. A comparable Trp damage in an argon saturated medium is due to the presence of large excess of H atoms along with OH radicals which can more eectively damage the Trp residues, even if the D50 is much lower (11 Gy). Rate constants for Tyr with OH radicals (1.3 1010 dm3 molÿ1 sÿ1) (Solar et al., 1984b; Geto, 1992) and with H atoms (4 108 dm3 molÿ1 sÿ1) (Anbar and Neta, 1967; Buxton et al., 1988) clearly justi®es the greater extent of Tyr damage in N2O saturated medium (Table 2). Comparable Tyr damage in argon and aerated medium may be due to a greater radical interaction with Tyr due to a much larger exposure (55 Gy) in an aerated medium, whereas in argon saturated condition the eect of H atoms together with OH radicals being 2
D50 is the dose required for 50% inactivation/damage.
much lethal even if the dose is as low as 11 Gy. Thus the eciency of OH radicals in initiating damage to the aromatics fall in line with that observed in other LDH studies, the only exception being ¯avocytochrome b2 do not show any protection for both residues after a threshold dose as observed in LDH (Winstead and Reece, 1970; Schuessler and Denkl, 1972). It is important to note that though OH radical is more damaging (as evidenced from the results in N2O saturated solution), its reactivity in aerated medium seems to be greatly reduced. This has, however, been explained earlier (Bhattacharyya et al., 1995) by assuming that molecular oxygen protects the enzyme from residue damage by oxygen ®xation process by which it prevents radical migration from initial site of damage to the potential site of damage. The re-appearance of the Tyr band in the denatured enzyme which was conspicuously absent in the native state for an lexc 280 nm may be ascribed to a partial energy transfer from Tyr to the Trp residues, a salient feature observed in the other LDH too (Hennessey and Johnson, 1981). Now it is known that amino acids spaced three and four apart in the linear sequence are spatially quite close to one another in an a-helix whereas those placed two apart are situated on the opposite sides of the helix and are less likely to make contact (Stryer, 1988). Flavocytochrome b2 essentially
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
75
has a helical structure. Hence in the two segments along the poly-peptide chain, between Tyr 144 and Trp 141, also between Tyr 254 and Trp 250 facile energy transfer can take place due to the close proximity between the donor and acceptor molecules. 3.2. Second derivative absorption spectrophotometry The second derivative absorption spectrophotometric technique was utilized to understand the extent of Tyr damage in the dierent media and relate it to the results obtained from ¯uorescence emission studies. The second derivative trough at 283 nm due to Tyr, though had interference from Trp but not from ¯avin. The second derivative absorption spectrum for Nacetyl tyrosinamide shows a trough at 283 nm (Fig. 3I) and that with it and the enzyme is shown in Fig. 3II. After correction for the contribution from Trp, second derivative absorbance value (d2A/dl 2)Tyr was plotted against CTyr/Cprotein for native (Fig. 4a) as well as the irradiated enzymes. The number of Tyr residues in the native enzyme (vide Fig. 4A) was in good agreement with the results obtained from an amino acid analyser (Lederer et al., 1985; Guiard, 1985). Table 2 records the number of Tyr residues remaining intact after irradiation at D50 for each of the three conditions which is found to be congruent with the results obtained from ¯uorescence emission studies. Here again the greater contribution of OH radicals in comparison to H atoms in initiating damage can be observed because lesser number of Tyr residues remained intact in a N2O saturated condition. Fig. 2. Changes in the Trp ¯uorescence emission (A) and Tyr ¯uorescence emission (B) (arbitrary units) on g-irradiation of ¯avocytochrome b2 under aerated (w), argon saturated (q) and N2O saturated conditions (r). The intensity for Trp ¯uorescence emission was measured at 350 nm for lexc at 297 nm and that for Tyr ¯uorescence emission was measured at 310 nm for lexc at 280 nm after adding 6 mol dmÿ3 GdmCl and 2 ME. 2.2 10ÿ6 mol dmÿ3 enzyme sample was taken in 0.1 mol dmÿ3 phosphate buer. y-axis in both A and B are in the `log' scale.
3.3. Quantum yields of Trp and Tyr residues The quantum yield values (Table 3) of Tyr both in native and irradiated (to an exposure of 6 Gy) state is considerably similar and the value is much less than that in its free state. A greater value in N2O saturated condition may be attributed to the greater eciency of OH radicals in increasing the deactivating pathways in
Table 2 Number of Tyr residue in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in neutral phosphate buer after irradiating at D50 doses for each of three conditions (aerated: 55 Gy, argon: 11 Gy and N2O: 28 Gy)a Number of Tyr residues remaining intact Irradiation conditions
Fluorescence emission
Second derivative absorption
Native Aerated Argon saturated Nitrous oxide saturated
16 14 14 8
16 14 13 8
a
Experimental uncertainty:212%. All results are an average of at least three sets of experiments.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Fig. 3. Second derivative absorption spectra of (i) N-Ac tyrosinamide in 6 mol dmÿ3 GdmCl and 2 ME in 0.1 mol dmÿ3 phosphate buer. The concentration of tyrosinamide were (a) 76.0 10ÿ6 mol dmÿ3 (b) 93.7 10ÿ6 mol dmÿ3 (c) 110.1 10ÿ6 mol dmÿ3 (d) 123.0 10ÿ6 mol dmÿ3 (e) 141.0 10ÿ6 mol dmÿ3 and (ii) unirradiated sample of ¯avocytochrome b2 titrated with N-acetyl tyrosinamide in 6 mol dmÿ3 GdmCl and 2 ME in 0.1 mol dmÿ3 phosphate buer (pH 7.0) where (a) 2.2 10ÿ6 mol dmÿ3 enzyme while the rest are the titration curves with CTyr/Cprot values for them being (b) 6.62 102 mol dmÿ3 (c) 3.29 102 mol dmÿ3 (d) 4.8 102 mol dmÿ3 and e) 6.962 102 mol dmÿ3.
the relaxation processes of Tyr. On denaturation, ff for tyrosine in both unirradiated and irradiated states approaches that of free Tyr indicating the exposure of the residues to the polar phase. Trp residues too showed no change from its native state for enzymes irradiated at a dose of 6 Gy. Quantum yields actually is the ratio of the ¯uorescence rate constant (kf ) and the sum of the rate constants of all the relaxation pathways inclusive of kf . But kf for Trp remains nearly constant, hence no new deactivating pathways emerge upon irradiation and the Trp residues are somehow protected from the aqueous phase with parity in the ff values for l280 and l297. However
on denaturation, all the Trp residues comes in contact with the polar phase, fF value matching with that of free Trp, corroborated by the red shift for lmax from ¯uorescence emission studies. 3.4. Time resolved studies Time resolved studies for total Trp emission intensity of the native, irradiated and denatured enzyme conforms to bi-exponential decay as adjudged by w 2 minimization. Though hti for native and irradiated (for a dose of 6 Gy) enzymes are by and large compar-
Table 3 Quantum yield values (ff ) for Trp and Tyr residues in neutral phosphate buer for native, irradiated and denatured ¯avocytochrome b2 (2.2 10ÿ6 mol dmÿ3)a Irradiation conditions
ff Trp (corrected) Exc. at 297 nm
ff Trp (corrected) Exc. at 280 nm
ff Tyr (corrected) Exc. at 280 nm
Native Aerated Argon saturated N2O saturated Denatured in 6 mol dmÿ3 GdmCl
0.117 0.118 0.119 0.119 0.149
0.110 0.118 0.110 0.126 0.145
0.068 0.072 0.066 0.084 0.117
a
All the results are an average of three to ®ve sets of experiments. Estimated error: 210%.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
able (Table 4), but for the denatured enzyme the mean lifetime approaches that of free Trp. This corroborates the nature of the results obtained from the quantum yield studies indicating the irradiation of 6 Gy does not cause any changes in the local micro-environment
77
of these residues, since the residues located in the hydrophobic interior are not exposed to the aqueous phase. 3.5. CD spectroscopic studies CD spectra of native and irradiated enzyme monitored in the far UV region (200±250 nm) does not show any perceptible changes in the helicity when irradiated in the range of 6 Gy (data not shown). But increasing the dose to 30 Gy shows a remarkable decrease in the helix content in the enzyme secondary structure irradiated in N2O and argon saturated condition, though no evolution towards a dominant b pleated sheet or a random coil emerge (Fig. 4b). The helicity is highly reduced in the N2O saturated condition indicating that interaction of OH radicals with the polypeptide chain triggers a more ecient damage in comparison to that with H atoms. Insigni®cant changes in an aerated condition may be due to fact that a dose of 30 Gy is far short of its D50 value (55 Gy) which actually inactivated the enzyme considerably and may thus lead to some structural changes or modi®cations, whereas the incident radiation being greater than D50 in each of the other two cases result in a conspicuous decrease in its inherent helicity. 3.6. Fluorescence quenching studies Trp ¯uorescence quenching studies were performed with native and irradiated enzyme solution, using both neutral quencher, acrylamide and charged ones, like
Fig. 4. (a). Plot of second derivative absorption values of Tyr residues in unirradiated ¯avocytochrome b2 against NTyr (CTyr/Cprot added). The intercept on the NTyr axis gives the measure of the Tyr residues in the enzyme. (b). Circular dichroism of ¯avocytochrome b2 in neutral 0.1 mol dmÿ3 phosphate buer for (a) native enzyme and those irradiated under (b) aerated (c) argon saturated and (d) nitrous oxide saturated conditions at a dose of 30 Gy for all the three irradiating conditions.
Fig. 5. Modi®ed Stern±Volmer plots for the quenching of Trp ¯uorescence of ¯avocytochrome b2 under (*) unirradiated, (w) aerated, (q) argon saturated and (r) N2O saturated condition by iodide ions. For the irradiated samples KI was added after irradiation at a dose of 30 Gy.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Table 4 Mean ¯uorescence lifetime data for Trp in native, irradiated and denatured 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in neutral phosphate buer with an excitation at 297 nm. A bi-exponential ®t was used for all the samplesa Irradiation conditions
t1, ns
a1
t2, ns
a2
hti
Native Aerated Argon saturated Nitrous oxide saturated Denatured in 6 mol dmÿ3 GdmCl
0.71 0.86 0.85 0.76 0.85
0.29 0.295 0.289 0.29 0.386
4.74 4.77 4.63 4.51 3.86
0.705 0.702 0.711 0.705 0.614
3.55 3.60 3.54 3.41 2.69
a
Estimated error: 210%.
iodide ions to have a better understanding of the nature of conformational changes. Since some Trp exists in the hydrophobic interior of the enzyme, the modi®ed Stern±Volmer equation I0 =DI 1= fa KQ Q 1=fa is used (Lehrer, 1971; Cantor and Schimmel, 1980); here fa is the fractional accessibility of the ¯uorophores, KQ is the Stern±Volmer constant and [Q] the quencher concentration. DI is the increment (I0-I ) in the ¯uorescence emission intensity due to the increased accessibility of the ¯uorophores to the quencher ions, compared to its native emission intensity. An incident radiation of 6 Gy fails to register any signi®cant changes in the ¯uorophore accessibility. Since signi®cant changes in the enzyme helicity were observed from circular dichroic studies at an exposure of 30 Gy, quenching studies were also performed by irradiating the enzyme at 30 Gy (Fig.5 and Table 5). Aerated condition did not show a very remarkable enhancement in Trp accessibility. However for N2O and argon saturated conditions whose D50 value is lower than 30 Gy did show a remarkable increase, larger being in N2O. Though no gross changes in the peak positions in our ¯uorescence emission studies were encountered, however, at an incident dose of 30 Gy, the accessibility of the quencher ions increased. Again the accessibility of dierent quenchers, e.g., acrylamide and iodide ions varied, iodide being smaller was found to be more accessible to the Trp ¯uoro-
phores. This clearly indicates that the polypeptide chain does not undergo a regular unfolding rather radiation results in breaking down the crucial H bonds in dierent pockets. H bond between -NH and -CO groups stabilizes the a-helix and breakdown of these H-bonds results in a wide-scale destruction of the peptide chains in dierent isolated pockets and not the whole chain in particular. As a result the dierent quenchers ®nd dierent accessibility to the ¯uorophores while the basic backbone is conserved with only a reduction in the percentage of helicity. An increase in the ¯uorophore accessibility with increasing radiation does not give rise to a simultaneous red shift of the Trp peak, indicating the microenvironment around these residues do not undergo changes that would originate from a gross unfolding of the main chain. Therefore, the decreased helicity results from the disruptions of the protein chain by way of breakage of some non-covalent bonds. Also modi®ed amino acid residues (at D50 dose) along the protein chain can create clefts and crevices and thus expose the hydrophobic regions in the enzyme to the aqueous phase without unfolding the secondary structure as a whole. It will be relevant to recall that radiation dose lesser than 6 Gy inactivated the enzyme (Bhattacharya et al., 1995) as monitored by the activity changes. Since inactivation preceded noticeable conformational changes it indicates that the enzyme active site is far more ¯exible and easily perturbable by the primary water radicals
Table 5 Fractional accessibilities ( fa) by Stern±Volmer quenching of Trp residues by iodide and acrylamide quenchers in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2, irradiating at a dose of 30 Gya Irradiating conditions
fa 100 value (by iodide quenchers)
fa 100 value (by acrylamide quenchers)
Unirradiated Air Argon saturated Nitrous oxide saturated
33.58 46.77 62.64 72.67
29.42 38.79 54.42 62.82
a
Experimental uncertainty: 28%. All results are an average of at least three sets of experiments.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
when compared to the rest of the protein structure. Since radiation primarily results in bond breakage, therefore these water radicals can have an easy access to the crucial active/binding site while the enzyme main chain remains unperturbed. It was also seen that the enzyme activity was much more sensitive to the presence of H atoms whereas our present study indicates a much greater extent of Trp and Tyr residue damage by OH radicals. OH radicals have a relatively high rate constants with these residues, hence it results in greater destruction of these residues. However since OH radicals are relatively unspeci®c, it tends to react less unevenly with the dierent active site residues. It also knocks out the stabilizing bonds and interactions in a random fashion and has a far more telling eect on the enzyme secondary structure than that initiated by the attack of H atoms. H atoms on the other hand being less reactive, have a better chance to penetrate the active site before reacting. Because of its selective nature, it can readily attack the crucial active site amino acid residues, thereby readily inactivating the enzyme.
Acknowledgements The authors would like to thank Professor S. K. Ghosh of Crystallography and Molecular Biology division for making available the CD spectropolarimeter. Also thanks are due to Professor Soumen Basak who took part in several fruitful discussions and gave some invaluable suggestions.
References Anbar, N., Neta, P., 1967. A compilation of speci®c bimolecular rate constants for the reaction of hydrated electrons, hydrogen atoms and hydroxyl radicals with inorganic and organic compound in aqueous solution. Int. J. Appl. Radiat. Isot. 18, 493. Balme, A., Lederer, F., 1994. On the rate of proton exchange with solvent of the catalytic histidine in ¯avocytochrome b2 (Yeast L-lactate dehydrogenase). Protein Sci. 3, 109. Bhattacharya, D., Saha, A., Mandal, P.C., 1995. Radiation induced inactivation of ¯avocytochrome b2 in dilute aqueous solution. Biochim. Biophys. Acta 1250, 1. Bielski, B.J.J., Cabelli, D.E., Arudi, R.L., 1985. Reactivity of HO2 /Oÿ 2 radicals in aqueous solution. J. Phys. Chem. Ref. Data 14, 1041. Buchanan, J.D., Armstrong, D.A., 1976. Free radical inactivation of lactate dehydrogenase. Int. J. Radiat. Biol. 36, 115. Buxton, G.V., Greenstock, C.L., Helman, W.P., Ross, A.B., 1988. Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals
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(OH /O ÿ) in aqueous solutions. J. Phys. Chem. Ref. Data 17, 513. Buxton, G.V., 1987. In: Farhataziz, Rodgers, M.A.J. (Eds.), Radiation Chemistry: Principles and Applications. VCH, New York, p. 321. Cantor, C.R., Schimmel, P.R., 1980. Biophysical Chemistry, Vol. II. Freeman, San Francisco, p. 409. Chapman, S.K., White, S.A., Reid, G.A., 1991. Flavocytochrome b2. Adv. Inorg. Chem. 36, 257. Geto, N., 1992. Pulse radiolysis of aromatic amino acids± State of the art. Amino Acids 2, 195. Guiard, B., 1985. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L (+)-lactate cytochrome c oxidireductase (cytochrome b2). EMBO J 4, 3265. Hennessey, J.P., Johnson, W.C., 1981. Information content in the circular dichroism of proteins. Biochemistry 20, 1085. Kirby, E.P., Steiner, R.F., 1970. The tryptophan microenvironment in apomyoglobin. J. Biol. Chem. 6300, 245. Lakowicz, J.R., 1983. Principles of Fluorescence Spectroscopy. Plenum Press, New York, p. 341. Lederer, F., 1974. On the ®rst steps of lactate oxidation by Baker's Yeast L-(+)-lactate dehydrogenase (cytochrome b2). Eur. J. Biochem. 46, 393. Lederer, F., Cortial, S., Becam, A.M., Haumont, P.Y., Perez, L., 1985. Complete amino acid sequence of ¯avocytochrome b2 from baker's yeast. Eur. J. Biochem. 152, 419. Lehrer, S.S., 1971. Solute perturbation of protein ¯uorescence. The quenching of the tryptophan ¯uorescence of model compounds and of lysozyme by iodide ions. Biochemistry 10, 3254. Miles, C.S., Rouviere-Fourmy, N., Lederer, F., Mathews, F.S., Reid, G.A., Chapman, S.K., 1992. Tyr-143 facilitates interdomain electron transfer in ¯avocytochrome b2. Biochem. J. 285, 187. Nozaki, Y., 1990. Determination of tryptophan, tyrosine and phenylalanine by second derivative spectrphotometry. Archs. Biochem. Biophys. 277, 324. Pajot, P., Claisse, M., 1974. Utilization by yeast of D-lactate and L-lactate as sources of energy in the presence of antimycin A. Eur. J. Biochem. 49, 275. Pinho Melo, E., Costa, S.M.B., Cabral, J.M.S., 1996. Denaturation of a recombinant cutinase from Fusarium solani in AOT-iso-octane reverse micelles Ð a steady state ¯uorescence study. Photochem. Photobiol. 63, 169. Rouviere-Fourmy, N., Capeillere-Blandin, C., Lederer, F., 1994. Role of tyrosine 143 in lactate dehydrogenation by ¯avocytochrome b2. Primary kinetic isotope eect studies with a phenylalanine mutant. Biochemistry 33, 798. Saha, A., Mandal, P.C., Bhattacharyya, S.N., 1993. Tyrosine residues in unirradiated and g-irradiated dihydro orotate dehydrogenase in dilute aqueous solution. Int. J. Radiat. Biol. 63, 557. Schuessler, H., Denkl, P., X-ray inactivation of lactate dehydrogenase in dilute solution, Int. J. Radiat. Biol. 21 435. Schuessler, H., Niemczyk, P., Eichhorn, M., Pauly, H., 1975. On the radiation induced aggregates of lactate dehydrogenase. Int. J. Radiat. Biol. 28, 401. Sharp, R.E., White, P., Chapman, S.K., Reid, G.A., 1994. Role of interdomain hinge of ¯avocytochrome b2 in intra and interprotein electron transfer. Biochemistry 33, 5115.
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Silvestrini, M.C., Tegoni, M., Celerier, J., Desbois, A., Gervais, M., 1993. Biochem. J. 295, 501. Solar, S., Solar, W., Geto, N., 1984a. Resolved multisite OH-attack on aqueous tryptophan studied by pulse radiolysis. Radiat. Phys. Chem. 23, 371. Solar, S., Solar, W., Geto, N., 1984b. Reactivity of OH with tyrosine in aqueous solution studied by pulse radiolysis. J. Phys. Chem. 88, 2091. Spinks, J.W.T., Woods, R.J., 1976. An Introduction to Radiation Chemistry, 2nd ed. Wiley, New York. Stryer, L., 1988. Biochemistry. Freeman, New York. White, P., Manson, F.D.C., Brunt, C.E., Chapman, S.K.,
Reid, G.A., 1993. The importance of the interdomain hinge in intramolecular electron transfer in ¯avocytochrome b2. Biochem. J. 291, 89. Winstead, J.A., Reece, J.C., 1970. Eects of gamma radiation in the chemical and physical properties of lactate dehydrogenase. Radiat. Res. 41, 125. Xia, Z.X., Mathews, F.S., 1990. Molecular structure of ¯avocytochrome b2 at 2.4 AÊ resolution. J. Mol. Biol. 212, 837. ÿ Ye, M., Schuler, R.H., 1986. The reaction of eÿ aq with H2PO4 as a source of hydrogen atom for pulse radiolysis studies in neutral solutions. Radiat. Phys. Chem. 28, 223.
www.elsevier.com/locate/radphyschem
Radiation induced modi®cation of tryptophan and tyrosine residues in ¯avocytochrome b2 in dilute aqueous solution D. Bhattacharya, A. Saha, P.C. Mandal* Nuclear Chemistry Division, Saha Institute of Nuclear Physics, 1/AF Bidhan nagar, Calcutta, 700 064, India Received 12 August 1999; accepted 10 November 1999
Abstract Steady state gamma irradiation of an aqueous solution of ¯avocytochrome b2 under dierent conditions led to modi®cation of tryptophan and tyrosine residues. These aromatic amino acid residues were more susceptible to the attack by OH radicals than H atoms. Unchanged quantum yield values for tryptophan and tyrosine residues and unchanged tryptophan excited state lifetime in the irradiated enzyme suggests that irradiation results in breakage of some non-covalent bonds disrupting the peptide framework partially. It is justi®ed by the circular dichroic studies for the irradiated enzyme which shows a reduced helicity but no evolution towards any other structures. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Flavocytochrome b2; Tryptophan; Tyrosine; Fluorescence quenching
1. Introduction Flavocytochrome b2, an integral member of the alternative respiratory chain in yeast (Pajot and Claisse, 1974) has been the centre of a ¯ourishing area of research (Miles et al., 1992; Silvestrini et al., 1993; White et al., 1993; Rouviere-Fourmy et al., 1994; Sharp et al., 1994). Characterization of its crystal structure (Xia and Mathews, 1990) at 0.24 nm resolution made it possible in unraveling of its catalytic, bio-physical and molecular biological aspects in ®ner details (Chapman et al., 1991; Balme and Lederer, 1994). However the eects of ionizing radiation on these systems remain an open question. Flavocytochrome b2, ideally belongs to the group of
* Corresponding author. Tel. +91-033-3370605; fax: +091033-3374637. E-mail address: [email protected] (P.C. Mandal).
lactate dehydrogenases (LDH) with wide digressions in structural aspects. Radiation-induced primary water radicals are known to attack at dierent sites in the enzyme. LDH, in fact, were found to undergo residue modi®cations and conformational changes which was believed to trigger due to radiation induced peptide chain breakage (Winstead and Reece, 1970; Schuessler and Denkl, 1972; Schuessler et al., 1975; Buchanan and Armstrong, 1976). It would be reasonable to anticipate that a signi®cant contribution of some speci®c reactions of this type may result in residue destruction or modi®cation which in turn might lead to changes in the enzyme secondary structure. The current work aims at elucidating the same in the ¯avin and hemelinked LDH, ¯avocytochrome b2 and relate it to the radiolytic responses observed in other LDH molecules. Modi®cations of tryptophan and tyrosine residues in enzymes irradiated with g-rays under dierent conditions were measured by change in ¯uorescence of tryptophan and tyrosine and also from second deriva-
0969-806X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 9 9 ) 0 0 5 2 0 - 4
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
tive absorption measurements. Any change in the conformations of the enzyme after irradiation was ascertained by CD studies.
2. Materials and Methods 2.1. Chemical Flavocytochrome b2 (from Saccharomyces cerevisiae ) E.C. 1.1.2.3, FMN, N-acetyl tyrosinamide and guanidinium chloride (GdmCl) were purchased from Sigma Chemical Co., USA. Phosphates and b-mercapto ethanol (2 ME) were of analar grade. 2.2. Desalting of the enzyme solution The required amount of enzyme suspension in 3.2 mol dmÿ3 ammonium sulphate solution was drawn out carefully and centrifuged. The precipitate thus obtained was dissolved in neutral 0.1 mol dmÿ3 phosphate buer after washing it twice in 70% ammonium sulphate (Lederer, 1974) was subsequently irradiated.
which otherwise tends to shift due to interfering contributions from Trp at this wavelength, thus making the analysis virtually impossible. The contributions of added Tyr in the form of N-acetyl tyrosinamide were taken into account during the calculation. The measurement was made using a Shimadzu 2101 spectrophotometer, the automatic correction mode was used routinely and the other settings are: slit set at 0.2 nm and the scan speed slow. 2.6. Quantum yields The quantum yields f, for Trp and Tyr were obtained by excitation at 280 nm or 297 nm, by using the relation, AE =AR fE ODE n2R =fR ODR n2E where A is the area under the curve, OD is the optical density, n is the refractive index, subscripts E and R refer to enzyme and reference substances respectively, free Trp and free Tyr were used as reference in neutral, phosphate buer, f values for both being 0.14 (Cantor and Schimmel, 1980; Pinho Melo et al., 1996).
2.3. Irradiation Procedure A 60Co g-source with an absorbed dose rate of 5.5 Gy/min determined by Fricke dosimeter (Spinks and Woods, 1976) was used, for irradiating the enzyme solution. 2.4. Steady state ¯uorescence measurements The ¯uorescence emission spectra were recorded with a Hitachi spectro¯uorimeter with entrance and exit slits set at 5 nm. The ¯uorescence emission intensity of tryptophan (Trp) only for excitation at 280 nm was obtained by normalization of the spectrum of lexc (297 nm) at 380 nm relative to that of lexc (280 nm) at 380 nm. Subsequent subtraction of the former from the latter gave the ¯uorescence emission due to tyrosine (Tyr) only (Pinho Melo et al., 1996). 2.5. Second derivative absorption studies The second derivative absorbance at a particular wavelength is the algebraic sum of the contributions of dierent components at that wavelength only. The technique employed here aims at determining the vertical distance from the baseline to the second derivative curve (Nozaki, 1990) at a wavelength assigned to a particular amino acid (283 nm for Tyr). N-acetyl tyrosinamide was used as a titrant that helps to retain the constancy of the trough position at 283 nm due to Tyr
2.7. Lifetime measurements The single photon counting technique was used to study the total intensity decays. The instrument was set up in our laboratory using the components supplied by Edinburgh Instruments (model 199F time domain ¯uorimeter). The decay pro®les of Trp in native, irradiated and denatured enzymes were obtained for 2500 photon counts at lem (335 nm) for lexc (297 nm). Lifetimes were estimated by ®tting each individual pro®le to the sum of two exponential decays and the accepted values correspond to the minimum w 2. Mean lifetimes were calculated by hti=a1t1+a2t2 where a is the fractional amplitude of the decay component with lifetime t. 2.8. Circular dichroic studies CD spectra for native and irradiated enzyme solutions were collected using a Jasco J-700 spectropolarimeter in quartz cells of 1 mm path length in the far UV range (200±250 nm). Five measurements were made for each sample at a scan speed of 20 nm/min, 2 s response time and step resolution of 0.1 nm. The spectra were corrected for the baseline by subtraction of the contribution due to blank solution (buer free from enzyme). The data were expressed in terms of mean residue ellipticity as deg cm2 dmolÿ1.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
3. Results and discussion Akin to biological systems, radiation induced damages of ¯avocytochrome b2 were studied in a dilute aqueous environment (0.1 mol dmÿ3 phosphate buer, pH 7.0). The high energy radiation is absorbed by the solvent, in this case, with the production of a number of primary water-derived radicals, namely hydrated electrons (eÿ aq), H atoms and OH radicals. In the absence of oxygen, each individual species has a characteristic eciency of its own such that the eciency of ¯uorophore loss can be designated as (Lakowicz, 1983) G -Fluorophore Sfi gi
1
where fi is the fractional eciency of the residue modi®cation and G1 (-Fluorophore) indicates the yield of the ¯uorophore loss under the same irradiation condition while gi is the primary yield of the respective radicals. In argon and N2O saturated conditions, according to Eq. (1) G(-Fluorophore) would be due to contribution from all the three radicals derived upon g-irradiation of an aqueous solution such that, G -Fluorophore fOH gOH fH gH feÿaq geÿaq
2
In N2O saturated medium, however, eÿ aq is converted to an equivalent amount of OH radicals, ÿ eÿ aq N2 O 4 OH OH N2
k 0:91 1010
dm3 molÿ1 sÿ1 Buxton et al:, 1988
3
such that Eq. (2) boils down to G(-Fluorophore)=fOHgOH '+fHgH gOH ' being the total yield of OH radicals indicating that there is an abundance of OH radicals in an N2O saturated medium. Since the studies are carried out in 0.1 mol dmÿ3 phosphate buer, hence in an argon saturated medium ÿ 2ÿ eÿ aq H2 PO4 4 H HPO4
k 1:9 107 dm3
molÿ1 sÿ1 Ye and Schuler, 1986
4
such that there is a predominance of H atoms, G -Fluorophore fOH gOH fH gH 0
5
where gH ' is the sum total of gH and geÿaq. 3.1. Fluorimetric studies To assess and quantitate the extent of damage of
1
G-unit=0.1034 mmol/J.
73
Trp and the Tyr residues after irradiation of ¯avocytochrome b2 ¯uorescence emission of the enzyme was followed before and after irradiation in 0.1 mol dmÿ3 phosphate buer with lex(297 nm) and lex (280 nm) (Fig. 1). The lmax observed at 335 nm (Fig. 1A(a)) is entirely due to the Trp residues in the enzyme which is a characteristic feature of a protein with a large number of Trp residues, the blue shift from that of free Trp (lmax 350 nm) is due to the shielding of the Trp residues from water molecules by the protein matrix. Interestingly irradiating the enzyme up to a dose of 12 or 18 Gy (Fig. 1A and 1C respectively) does not indicate any change in the ¯uorescence emission spectrum in a N2O saturated medium, though a considerable reduction in the emission intensity occurs. An almost unchanged lmax for an irradiated enzyme indicates that practically no enhancement of polarity occurs. When the ¯uorescence emission of native enzyme was followed, after denaturing it in 6 mol dmÿ3 GdmCl and 2 ME and exciting at 280 nm lmax for emission registers a red shift (350 nm) with the appearance of another distinct peak at 310 nm due to the Tyr moieties in the enzyme (Fig. 1A(d)). Now, when the concentration of both the irradiated and the unirradiated samples are the same, it is possible to judge the extent of their damage from the ratio of the ¯uorescence intensities of the unirradiated sample to the irradiated one (Saha et al., 1993) both denatured in 6 mol dmÿ3 GdmCl and 2 ME. The ¯uorescence intensity of both Trp (Fig. 2A) and Tyr (Fig. 2B) residues in the irradiated samples (in 0.1 mol dmÿ3 phosphate buer, pH 7.0) under dierent conditions in the presence of the denaturant decreased exponentially with increasing dose indicating linear semi-log plots in Fig. 2. When irradiation is carried out in aerated medium, molecular oxygen converts H atoms and eÿ aq to HO2 ÿ and O2 radicals respectively (Buxton et al., 1988). Literature values (Buxton, 1987; Bielski et al., 1985) show these two radicals do not react with Trp and Tyr. Therefore OH radicals alone are responsible for
Table 1 Number of Trp residues in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 after irradiating at D50 for each of the three conditions (aerated: 55 Gy, argon: 11 Gy and N2O: 28 Gy)a Irradiation conditions
Number of Trp residues
Native Aerated Nitrous oxide saturated Argon saturated
5 4 3 4
a Experimental uncertainty: 210%. All results are an average of at least three sets of experiments.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Fig. 1. (A). Corrected and normalized spectra of 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in 0.1 mol dmÿ3 phosphate buer (pH 7.0). The four spectra depicted here represents both native and denatured enzymes. Spectra (a) and (b) represents that of native enzyme with an excitation at (a) 297 nm and (b) 280 nm. (c) and (d) represents the ¯uorescence emission spectrum of enzyme denatured in the presence of 6 mol dmÿ3 GdmCl and 2 ME with an excitation of (c) 297 nm and (d) 280 nm, (B) Fluorescence emission spectra (excitation at 297 nm) of ¯avocytochrome b2 in 0.1 mol dmÿ3 phosphate buer (pH 7.0) irradiated under N2O saturated condition at dierent doses in the absence of the denaturant, (C) same as in (B) but after denaturing in 6 mol dmÿ3 GdmCl in the presence of 2 ME.
the damage in this case. Rate constants of Trp at neutral pH with OH is 1.3 1010 dm3 molÿ1 sÿ1 (Solar et al.,1984a; Geto, 1992) whereas that with H atoms is 2 109 dm3 molÿ1 sÿ1 (Kirby and Steiner, 1970) explains the greater extent of residue damage in N2O saturated conditions (Table 1) where there is an abundance of OH radicals. Exposure to a very high dose (D502, 55 Gy) (Bhattacharya et al., 1995) in aerated medium possibly allows a greater interaction of the OH radicals with the Trp residues. A comparable Trp damage in an argon saturated medium is due to the presence of large excess of H atoms along with OH radicals which can more eectively damage the Trp residues, even if the D50 is much lower (11 Gy). Rate constants for Tyr with OH radicals (1.3 1010 dm3 molÿ1 sÿ1) (Solar et al., 1984b; Geto, 1992) and with H atoms (4 108 dm3 molÿ1 sÿ1) (Anbar and Neta, 1967; Buxton et al., 1988) clearly justi®es the greater extent of Tyr damage in N2O saturated medium (Table 2). Comparable Tyr damage in argon and aerated medium may be due to a greater radical interaction with Tyr due to a much larger exposure (55 Gy) in an aerated medium, whereas in argon saturated condition the eect of H atoms together with OH radicals being 2
D50 is the dose required for 50% inactivation/damage.
much lethal even if the dose is as low as 11 Gy. Thus the eciency of OH radicals in initiating damage to the aromatics fall in line with that observed in other LDH studies, the only exception being ¯avocytochrome b2 do not show any protection for both residues after a threshold dose as observed in LDH (Winstead and Reece, 1970; Schuessler and Denkl, 1972). It is important to note that though OH radical is more damaging (as evidenced from the results in N2O saturated solution), its reactivity in aerated medium seems to be greatly reduced. This has, however, been explained earlier (Bhattacharyya et al., 1995) by assuming that molecular oxygen protects the enzyme from residue damage by oxygen ®xation process by which it prevents radical migration from initial site of damage to the potential site of damage. The re-appearance of the Tyr band in the denatured enzyme which was conspicuously absent in the native state for an lexc 280 nm may be ascribed to a partial energy transfer from Tyr to the Trp residues, a salient feature observed in the other LDH too (Hennessey and Johnson, 1981). Now it is known that amino acids spaced three and four apart in the linear sequence are spatially quite close to one another in an a-helix whereas those placed two apart are situated on the opposite sides of the helix and are less likely to make contact (Stryer, 1988). Flavocytochrome b2 essentially
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
75
has a helical structure. Hence in the two segments along the poly-peptide chain, between Tyr 144 and Trp 141, also between Tyr 254 and Trp 250 facile energy transfer can take place due to the close proximity between the donor and acceptor molecules. 3.2. Second derivative absorption spectrophotometry The second derivative absorption spectrophotometric technique was utilized to understand the extent of Tyr damage in the dierent media and relate it to the results obtained from ¯uorescence emission studies. The second derivative trough at 283 nm due to Tyr, though had interference from Trp but not from ¯avin. The second derivative absorption spectrum for Nacetyl tyrosinamide shows a trough at 283 nm (Fig. 3I) and that with it and the enzyme is shown in Fig. 3II. After correction for the contribution from Trp, second derivative absorbance value (d2A/dl 2)Tyr was plotted against CTyr/Cprotein for native (Fig. 4a) as well as the irradiated enzymes. The number of Tyr residues in the native enzyme (vide Fig. 4A) was in good agreement with the results obtained from an amino acid analyser (Lederer et al., 1985; Guiard, 1985). Table 2 records the number of Tyr residues remaining intact after irradiation at D50 for each of the three conditions which is found to be congruent with the results obtained from ¯uorescence emission studies. Here again the greater contribution of OH radicals in comparison to H atoms in initiating damage can be observed because lesser number of Tyr residues remained intact in a N2O saturated condition. Fig. 2. Changes in the Trp ¯uorescence emission (A) and Tyr ¯uorescence emission (B) (arbitrary units) on g-irradiation of ¯avocytochrome b2 under aerated (w), argon saturated (q) and N2O saturated conditions (r). The intensity for Trp ¯uorescence emission was measured at 350 nm for lexc at 297 nm and that for Tyr ¯uorescence emission was measured at 310 nm for lexc at 280 nm after adding 6 mol dmÿ3 GdmCl and 2 ME. 2.2 10ÿ6 mol dmÿ3 enzyme sample was taken in 0.1 mol dmÿ3 phosphate buer. y-axis in both A and B are in the `log' scale.
3.3. Quantum yields of Trp and Tyr residues The quantum yield values (Table 3) of Tyr both in native and irradiated (to an exposure of 6 Gy) state is considerably similar and the value is much less than that in its free state. A greater value in N2O saturated condition may be attributed to the greater eciency of OH radicals in increasing the deactivating pathways in
Table 2 Number of Tyr residue in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in neutral phosphate buer after irradiating at D50 doses for each of three conditions (aerated: 55 Gy, argon: 11 Gy and N2O: 28 Gy)a Number of Tyr residues remaining intact Irradiation conditions
Fluorescence emission
Second derivative absorption
Native Aerated Argon saturated Nitrous oxide saturated
16 14 14 8
16 14 13 8
a
Experimental uncertainty:212%. All results are an average of at least three sets of experiments.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Fig. 3. Second derivative absorption spectra of (i) N-Ac tyrosinamide in 6 mol dmÿ3 GdmCl and 2 ME in 0.1 mol dmÿ3 phosphate buer. The concentration of tyrosinamide were (a) 76.0 10ÿ6 mol dmÿ3 (b) 93.7 10ÿ6 mol dmÿ3 (c) 110.1 10ÿ6 mol dmÿ3 (d) 123.0 10ÿ6 mol dmÿ3 (e) 141.0 10ÿ6 mol dmÿ3 and (ii) unirradiated sample of ¯avocytochrome b2 titrated with N-acetyl tyrosinamide in 6 mol dmÿ3 GdmCl and 2 ME in 0.1 mol dmÿ3 phosphate buer (pH 7.0) where (a) 2.2 10ÿ6 mol dmÿ3 enzyme while the rest are the titration curves with CTyr/Cprot values for them being (b) 6.62 102 mol dmÿ3 (c) 3.29 102 mol dmÿ3 (d) 4.8 102 mol dmÿ3 and e) 6.962 102 mol dmÿ3.
the relaxation processes of Tyr. On denaturation, ff for tyrosine in both unirradiated and irradiated states approaches that of free Tyr indicating the exposure of the residues to the polar phase. Trp residues too showed no change from its native state for enzymes irradiated at a dose of 6 Gy. Quantum yields actually is the ratio of the ¯uorescence rate constant (kf ) and the sum of the rate constants of all the relaxation pathways inclusive of kf . But kf for Trp remains nearly constant, hence no new deactivating pathways emerge upon irradiation and the Trp residues are somehow protected from the aqueous phase with parity in the ff values for l280 and l297. However
on denaturation, all the Trp residues comes in contact with the polar phase, fF value matching with that of free Trp, corroborated by the red shift for lmax from ¯uorescence emission studies. 3.4. Time resolved studies Time resolved studies for total Trp emission intensity of the native, irradiated and denatured enzyme conforms to bi-exponential decay as adjudged by w 2 minimization. Though hti for native and irradiated (for a dose of 6 Gy) enzymes are by and large compar-
Table 3 Quantum yield values (ff ) for Trp and Tyr residues in neutral phosphate buer for native, irradiated and denatured ¯avocytochrome b2 (2.2 10ÿ6 mol dmÿ3)a Irradiation conditions
ff Trp (corrected) Exc. at 297 nm
ff Trp (corrected) Exc. at 280 nm
ff Tyr (corrected) Exc. at 280 nm
Native Aerated Argon saturated N2O saturated Denatured in 6 mol dmÿ3 GdmCl
0.117 0.118 0.119 0.119 0.149
0.110 0.118 0.110 0.126 0.145
0.068 0.072 0.066 0.084 0.117
a
All the results are an average of three to ®ve sets of experiments. Estimated error: 210%.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
able (Table 4), but for the denatured enzyme the mean lifetime approaches that of free Trp. This corroborates the nature of the results obtained from the quantum yield studies indicating the irradiation of 6 Gy does not cause any changes in the local micro-environment
77
of these residues, since the residues located in the hydrophobic interior are not exposed to the aqueous phase. 3.5. CD spectroscopic studies CD spectra of native and irradiated enzyme monitored in the far UV region (200±250 nm) does not show any perceptible changes in the helicity when irradiated in the range of 6 Gy (data not shown). But increasing the dose to 30 Gy shows a remarkable decrease in the helix content in the enzyme secondary structure irradiated in N2O and argon saturated condition, though no evolution towards a dominant b pleated sheet or a random coil emerge (Fig. 4b). The helicity is highly reduced in the N2O saturated condition indicating that interaction of OH radicals with the polypeptide chain triggers a more ecient damage in comparison to that with H atoms. Insigni®cant changes in an aerated condition may be due to fact that a dose of 30 Gy is far short of its D50 value (55 Gy) which actually inactivated the enzyme considerably and may thus lead to some structural changes or modi®cations, whereas the incident radiation being greater than D50 in each of the other two cases result in a conspicuous decrease in its inherent helicity. 3.6. Fluorescence quenching studies Trp ¯uorescence quenching studies were performed with native and irradiated enzyme solution, using both neutral quencher, acrylamide and charged ones, like
Fig. 4. (a). Plot of second derivative absorption values of Tyr residues in unirradiated ¯avocytochrome b2 against NTyr (CTyr/Cprot added). The intercept on the NTyr axis gives the measure of the Tyr residues in the enzyme. (b). Circular dichroism of ¯avocytochrome b2 in neutral 0.1 mol dmÿ3 phosphate buer for (a) native enzyme and those irradiated under (b) aerated (c) argon saturated and (d) nitrous oxide saturated conditions at a dose of 30 Gy for all the three irradiating conditions.
Fig. 5. Modi®ed Stern±Volmer plots for the quenching of Trp ¯uorescence of ¯avocytochrome b2 under (*) unirradiated, (w) aerated, (q) argon saturated and (r) N2O saturated condition by iodide ions. For the irradiated samples KI was added after irradiation at a dose of 30 Gy.
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D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
Table 4 Mean ¯uorescence lifetime data for Trp in native, irradiated and denatured 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2 in neutral phosphate buer with an excitation at 297 nm. A bi-exponential ®t was used for all the samplesa Irradiation conditions
t1, ns
a1
t2, ns
a2
hti
Native Aerated Argon saturated Nitrous oxide saturated Denatured in 6 mol dmÿ3 GdmCl
0.71 0.86 0.85 0.76 0.85
0.29 0.295 0.289 0.29 0.386
4.74 4.77 4.63 4.51 3.86
0.705 0.702 0.711 0.705 0.614
3.55 3.60 3.54 3.41 2.69
a
Estimated error: 210%.
iodide ions to have a better understanding of the nature of conformational changes. Since some Trp exists in the hydrophobic interior of the enzyme, the modi®ed Stern±Volmer equation I0 =DI 1= fa KQ Q 1=fa is used (Lehrer, 1971; Cantor and Schimmel, 1980); here fa is the fractional accessibility of the ¯uorophores, KQ is the Stern±Volmer constant and [Q] the quencher concentration. DI is the increment (I0-I ) in the ¯uorescence emission intensity due to the increased accessibility of the ¯uorophores to the quencher ions, compared to its native emission intensity. An incident radiation of 6 Gy fails to register any signi®cant changes in the ¯uorophore accessibility. Since signi®cant changes in the enzyme helicity were observed from circular dichroic studies at an exposure of 30 Gy, quenching studies were also performed by irradiating the enzyme at 30 Gy (Fig.5 and Table 5). Aerated condition did not show a very remarkable enhancement in Trp accessibility. However for N2O and argon saturated conditions whose D50 value is lower than 30 Gy did show a remarkable increase, larger being in N2O. Though no gross changes in the peak positions in our ¯uorescence emission studies were encountered, however, at an incident dose of 30 Gy, the accessibility of the quencher ions increased. Again the accessibility of dierent quenchers, e.g., acrylamide and iodide ions varied, iodide being smaller was found to be more accessible to the Trp ¯uoro-
phores. This clearly indicates that the polypeptide chain does not undergo a regular unfolding rather radiation results in breaking down the crucial H bonds in dierent pockets. H bond between -NH and -CO groups stabilizes the a-helix and breakdown of these H-bonds results in a wide-scale destruction of the peptide chains in dierent isolated pockets and not the whole chain in particular. As a result the dierent quenchers ®nd dierent accessibility to the ¯uorophores while the basic backbone is conserved with only a reduction in the percentage of helicity. An increase in the ¯uorophore accessibility with increasing radiation does not give rise to a simultaneous red shift of the Trp peak, indicating the microenvironment around these residues do not undergo changes that would originate from a gross unfolding of the main chain. Therefore, the decreased helicity results from the disruptions of the protein chain by way of breakage of some non-covalent bonds. Also modi®ed amino acid residues (at D50 dose) along the protein chain can create clefts and crevices and thus expose the hydrophobic regions in the enzyme to the aqueous phase without unfolding the secondary structure as a whole. It will be relevant to recall that radiation dose lesser than 6 Gy inactivated the enzyme (Bhattacharya et al., 1995) as monitored by the activity changes. Since inactivation preceded noticeable conformational changes it indicates that the enzyme active site is far more ¯exible and easily perturbable by the primary water radicals
Table 5 Fractional accessibilities ( fa) by Stern±Volmer quenching of Trp residues by iodide and acrylamide quenchers in 2.2 10ÿ6 mol dmÿ3 ¯avocytochrome b2, irradiating at a dose of 30 Gya Irradiating conditions
fa 100 value (by iodide quenchers)
fa 100 value (by acrylamide quenchers)
Unirradiated Air Argon saturated Nitrous oxide saturated
33.58 46.77 62.64 72.67
29.42 38.79 54.42 62.82
a
Experimental uncertainty: 28%. All results are an average of at least three sets of experiments.
D. Bhattacharya et al. / Radiation Physics and Chemistry 59 (2000) 71±80
when compared to the rest of the protein structure. Since radiation primarily results in bond breakage, therefore these water radicals can have an easy access to the crucial active/binding site while the enzyme main chain remains unperturbed. It was also seen that the enzyme activity was much more sensitive to the presence of H atoms whereas our present study indicates a much greater extent of Trp and Tyr residue damage by OH radicals. OH radicals have a relatively high rate constants with these residues, hence it results in greater destruction of these residues. However since OH radicals are relatively unspeci®c, it tends to react less unevenly with the dierent active site residues. It also knocks out the stabilizing bonds and interactions in a random fashion and has a far more telling eect on the enzyme secondary structure than that initiated by the attack of H atoms. H atoms on the other hand being less reactive, have a better chance to penetrate the active site before reacting. Because of its selective nature, it can readily attack the crucial active site amino acid residues, thereby readily inactivating the enzyme.
Acknowledgements The authors would like to thank Professor S. K. Ghosh of Crystallography and Molecular Biology division for making available the CD spectropolarimeter. Also thanks are due to Professor Soumen Basak who took part in several fruitful discussions and gave some invaluable suggestions.
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