Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers

Biochimica et Biophysica Acta, 1203(1993) 11-17

11

© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

BBAPRO 34585

Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers Elisabeth Franzini, Hassan Sellak, Jacques Hakim and Catherine Pasquier INSERM U294, Facult~Xavier Bichat, Paris (France)

(Received 1 February 1993)

Key words: Lysozyme;Hydroxylradical; Radiolysis; Free radical The hydroxyl radical (OH') is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH' scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH'-induced alterations of a protein, lysozyme. Lysozyme was inactivated by OH' with a yield of 6.5 mol OH "/mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH" scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH" with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.

Introduction

The damaging effect of hydroxyl radical OH" on biological systems is mainly associated with its high reactivity with molecules at its site of formation [1-7]. This leads to protein denaturation and to the formation of secondary radicals, which can also cause damage or modify the effect of O H ", as previously reported for lysozyme [8-12]. During the oxidative burst, polymorphonuclear leukocytes (PMN) release activated oxygen species and enzymes such as lysozyme, that might interact [13], and that might be of importance in inflammatory conditions. O H attacks all amino acids but preferentially targets tryptophan, cysteine, histidine and tyrosine [1416]; as two tryptophans have been shown to be part of the active site of lysozyme lying close to /3-pleated sheets [17], their attack may lead to enzyme inactivation. On the other hand, protein attack by O H can induce tyrosyl radical formation, which in turn leads to aggregate formation that can be prevented by reduction of the tyrosyl radical [14]. Pentoxifylline (Ptx, 1-(5-oxohexyl)-3,7-dimethylxanthine) is a trisubstituted purine used in the treat-

Correspondence to: C. Pasquier, INSERM U294, Facult6 Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.

ment of intermittent claudication and peripheral vascular disease [18,19]. Ptx has been shown to be effective in reducing late radiation injury in mice [20] and in protecting animals from the deleterious effects of inflammatory reactions [21,22] which are linked, in part, to O H ' toxicity. Moreover, we and others have shown that Ptx, uric acid and thymine react with OH" very rapidly and give both oxidizing and reducing radicals [23-26] which are deleterious and or protective, respectively. The aim of this study was to determine whether O H scavenging by Ptx, thymine or uric acid protected lysozyme by acting on particular amino acids of the active sites of the enzymes, e.g., tryptophan and tyrosine, through formation of reducing radicals [10]. We therefore subjected lysozyme to attack by OH" produced by gamma radiolysis in the presence and absence of scavengers, and subsequently analyzed its activity and structure, as well as its tryptophan and tyrosine content. Materials and Methods Materials. Pentoxifylline (1-(5-oxo-hexyl)-3,7-dimethylxanthine, Trental, purity 99.3%) was a generous gift from Hoechst (Paris, France); phosphate buffer ( N a H 2 P O 4, 2 H 2 0 ) ( N o r m a p u r ) w a s from Rh6ne-Poulenc (Paris, France); uric acid, thymine, grade III

12 lysozyme from chicken egg-white, N , N , N ',N '-tetramethylethylenediamine (TEMED), ammonium persulfate, bovine serum albumin (BSA), lyophilized Micrococcus lysodeikticus, Coomassie brillant blue, and Ltryptophan were from Sigma (St. Louis, MO, USA); N20 (purity > 99.99%) was from Compagnie Fran~aise des Produits Oxyg6n6s (Meudon, France); KH2PO4, NaCI and urea were from Merck (Darmstadt, Germany); acrylamide, bis-acrylamide, glycine, SDS, Tris and the silver-stain kit were from Bio-Rad (Richmond, CA, USA); the low-molecular-weight marker kit was from Pierce (Rockford, IL, USA); /3-mercaptoethanol and Sephadex G-75 were from Pharmacia (Uppsala, Sweden). 2,2'-biphenol was from Aldrich (Steinheim, Germany). Water and glassware used in gamma radiolysis were treated as described in Ref. 23. Gamma radiolysis. Hydroxyl radicals were produced by steady-state irradiation of saturated N=O solutions in a 6°Co irradiator as previously described [23]. The intensity of the source was 0.33 Gy/s, giving a O H production of 0.18/zM/s. Lysozyme, alone or in combination with scavengers, was irradiated at room temperature in 1 ml of 10 mM phosphate buffer (PB-N20 (pH 7.4)) and then stored at -20°C. The same, nonirradiated solutions bubbled with N20 served as controis. After thawing, lysozyme activity was measured according to the method of Shugar [28], using a Uvikon 860 (Kontron Instruments, Zurich, Switzerland). Fluorescence measurements. Tryptophan and bityrosine residues in the treated lysozyme were quantified by means of fluorimetry. Fluorescence spectra were recorded on an Aminco SPF 500 spectrofluorimeter. All experimental solutions were made in 10 mM PB (pH 7.4) at 20°C. Tryptophan was measured in 6 M guanidine at the maximum of the fluorescence spectra emission at 360 nm (290 nm excitation wavelength)

10-~

--~ "O

6-

~

20£ 50

i 100

I

150

Dose (Gy) Fig. 1. Inactivation of lysozyme as a function of radiation dose. Decrease in lysozyme activity (expressed as /zM of degraded lysozyme) as a function of radiation dose (Gy) at three initial concentrations of lysozyme: (o), 3.4/zM; (ra), 6.9/zM and ( - ) , 10.6 /xM. (Each point represents the mean+S.E, of five experiments). The straight line represents the initial rate of lysozyme degradation as a function of the radiation dose.

[29]; native lysozyme was used to measure 100% fluorescence. For bityrosine measurements, the protein was denatured in 6 M urea and the emission was monitored at 410 nm (excitation at 315 nm). Calibration was done with biphenol in 6 M urea [30]. Polyacrylamide gel electrophoresis. Lysozyme was analysed by means of SDS-PAGE [31] with linear gradient resolving gels from 10 to 20% (w/v). The samples (1/zg of protein) were run reduced [31]; they were then fixed and stained with Coomassie brillant blue. After complete decoloration, gels were silver-stained. After each coloration, densitometry was performed with a Preference Sebia apparatus (Issy les Moulineaux, France). All the relevant parameters and a standard were established for each gel. Protein was detectable with a sensitivity of 0.1 ~g and a detection limit of 0.2 /xg. The method has a reproducibility of 5%. Gel filtration. 1 ml of protein fractions obtained after irradiation of 6.9/zM lysozyme solution, with or without scavenger, were separated by gel filtration on a Sephadex G-75 column (600 × 10 mm, 0.166 ml/min) by elution with phosphate buffer (0.05 M NaH2PO 4, 0.05 M K H 2 P O 4 , 0.154 M NaCI (pH 6.6)). Fractions of 1 ml each were collected and recorded at 280 nm. The column was calibrated with BSA (60 kDa), trypsin (24 kDa) and lysozyme (14.4 kDa). Statistical analysis. Results are given as mean _+ standard error of the mean (S.E.) of separate experiments. Data were compared using Student's t-test. Regression analysis was performed using an IBM personal computer. Results

Hydroxyl radical effects on lysozyme Lysozyme exposed to gamma irradiation (0 to 125 Gy) was inactivated with a yield of 6.5 mol OH '/mol lysozyme independently of its concentration (3.4, 6.9 or 10.6 /xM), as shown in Fig. 1. Addition of H 2 0 2 (5" 10-6-10-4M) to the lysozyme solution did not modify the yield of inactivation of the irradiated enzyme. Irradiation of lysozyme (6.9 /zM) from 0 to 60 Gy induced (i), a disappearance of the SDS-PAGE band at 14.4 kDa; (ii), an aggregation of lysozyme giving bands at 28 kDa and 42 kDa and (iii), the appearance of two new bands of 8 and 6.5 kDa (Fig. 2). Lysozyme activity and the band at 14.4 kDa decreased as a function of the irradiation dose (Fig. 3). The decrease in the lysozyme tryptophan fluorescence intensity (Fig. 4) was related to the irradiation dose (P < 0.001) and to the loss of lysozyme activity. At a dose of 55 Gy (54% inactivation), tryptophan fluorescence decreased by 44%. Bityrosine fluorescence was detectable above 20 Gy (Fig. 4). The loss of enzyme activity occurred before bityrosine production became detectable.

13

94 67

--

43 4"-'-30 20 17 14 8

6.2 2.5

A Dose

(Gy)

0

A

A

A

A

A

10

20

30

40

55

Fig. 2. SDS-PAGE of irradiated lysozyme. SDS-PAGE of lysozyme (6.9 ~M) exposed to various radiation doses (0-55 Gy) were silver-stained. The initial 14.4 kDa band gives rise, at 20 and 30 Gy, to fragments of 8 and 6.5 kDa and aggregates of 28 and 42 kDa.

Yields of scavenger degradation by O H

Protection of lysozyme by O H scavengers

N20-saturated aqueous solutions of Ptx, uric acid and thymine (10 to 200 ~M) were irradiated from 0 to 60 Gy. The resulting absorbance, measured at the respective absorption maxima (274 nm for Ptx, 265 nm for thymine and 290 nm for uric acid), decreased with increasing doses of irradiation, independent of the scavenger concentration. For Ptx and thymine, it is known that the oxidation product does not absorb (at 274 nm and 265 nm, respectively) [23,32]; it can thus be assumed that the oxidation of both molecules required two molecules of O H for one molecule of scavenger. The oxidation of uric acid gives allantoin which does not absorb at any wavelength [33,34]; however, other products could be formed from this oxidation, the absorbance of which is unknown. The yield could thus be less than 2.

The effects of the scavengers on lysozyme (6.9 ~ M) was tested in PB-N20 exposed to 55 Gy of gamma irradiation. In the absence of scavenger, 54% of lysozyme was inactivated (3.2 /xM remained active). Scavenger concentrations from 0 to 47 /zM did not alter lysozyme activity by themselves. Different patterns of protection were obtained (Fig. 5). Ptx and uric acid completely protected lysozyme at a concentration of 6.8 ~M; the protection obtained with thymine varied linearly with its concentration and was complete above 20/~ M. Thus, the optimal concentrations for complete protection were different for the three scavengers, although they all scavenged OH' with approximately the same high rate constants, i.e. 7.2.10 9 M -1 s - 1 , 7.7" 10 9 M -1 s -1, 5- 10 9 M -t s -t for uric acid, pentoxifylline and thymine, respectively [16,23-25]. Modifications of lysozyme were analysed by SDSPAGE following irradiation with 55 Gy in the presence of the scavengers in two conditions: first, in the presence of a large amount of scavengers leading to total

~

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50

=,. m

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i

o

i

20

40

i

0

*~

60

50 ~

*-. 0

•

20 E P

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Fig. 3. Decrease in lysozyme activity and its 14.4 kDa band as a function of radiation dose. Each point (e) of the 14.4 kDa band represents densitometric measurement after Coomassie staining; (mean+ S.E. of six experiments, r = 0.994, P < 0.001). The decrease in lysozyme activity (zx) is expressed as a percentage of residual lysozyme activity related to native lysozyme; each point represents the mean+S.E, of three or more experiments (6.9 ~ M lysozyme irradiated from 0 to 60 Gy).

,.I

0

0

0

40 20 Dose (Gy)

60

~ ..a

Fig. 4. Tryptophan and bityrosine fluorescence of lysozyme bityrosine formation as a function of the radiation dose. Fluorescence of tryptophan (e) and bityrosine ([]) of 6.9/.t M lysozyme are expressed as a function of the irradiation dose (Gy); the curves are representative of three different experiments.

14

7 .

.=

I

"~

",

5

o O.

0

10

20

30

S ~M

Fig. 5. Protection of lysozymeactivity by O H scavengers. Protected lysozyme is expressed in ~M as a function of the scavenger concentration ((O), uric acid; (e), Ptx; (r,), thymine). Each point represents the mean 5:S.E. of four experiments. The initial residual concentration of lysozymeirradiated by 55 Gy is 3.2/~M (y-axis).

protection of enzyme activity; second, at a scavenger concentration protecting 75% of the lysozyme activity (Table I). In the first condition, with Ptx and uric acid, the 14.4 kDa band represented 88%, and no aggregates or fragments were observed. In contrast, with thymine, only 44% of the 14.4 kDa band was preserved; the 28 kDa and 42 kDa aggregates represented 44 and 10%, respectively, and no small fragments were observed (Table I). At high concentrations, uric acid and Ptx apparently did not mediate radical-induced alterations, but were able to prevent O H "-induced enzyme inactivation and to protect secondary structure. In contrast, thymine radicals or derivatives appeared to alter lysozyme structure, leading to aggregate formation but no loss of enzyme activity. To obtain 75% residual lysozyme activity, lysozyme alone was irradiated with 30 Gy, whereas in presence of scavengers 55 Gy was required (Table I). Under these conditions, the 14.4 kDa band was less intense

with uric acid and Ptx (49 and 46% for uric acid and Ptx, respectively) than with lysozyme alone, suggesting that activity remained in bands other than 14.4 kDa. Fragmentation was greater with uric acid and Ptx than with lysozyme alone (21% and 34%, respectively). There was no fragmentation with thymine, but aggregation (28 and 42 kDa) was greater than with the other scavengers. Lysozyme (6.9 izM) irradiated at 55 Gy retained 56% of its initial tryptophan fluorescence (Table II). Regardless of the thymine or uric acid concentration, tryptophan fluorescence was in the range of 80%, whereas it increased as a function of the Ptx concentration, suggesting that Ptx was specifically protecting the active site represented by its two tryptophans [17]. Thymine and uric acid, although preserving lysozyme activity, did not seem to be specific for the active site, and probably reacted also with the other tryptophan residues of the molecule. Moreover, uric acid, which inhibited bityrosine fluorescence as a function of its concentration, and Ptx consistently, gave about the same total inhibition whatever its concentration. In contrast, bityrosine fluorescence increased with the thymine concentration, suggesting that aggregation of lysozyme was promoted. Since 50% of the 14.4 kDa band disappeared when thymine fully protected lysozyme activity, we looked for lysozyme activity in the other fractions corresponding to the 28 and 42 kDa bands after their separation by gel filtration. Nonirradiated lysozyme eluted from the Sephadex G-75 column as a protein peak in fraction 32 (yield 92%). Lysozyme irradiated with 30 Gy showed a protein peak in fraction 32 (14.4 kDa band) accounting for all the residual activity (75%). Samples irradiated in the presence of thymine gave three active peaks: fraction 13 (42 kDa aggregate), fraction 26 (28 kDa aggregate) and the 14.4 kDa native form (fraction 32). In this

TABLE I Scavenger effects on OH -induced fragmentation and aggregation of lysozyme

Lysozyme(6.9 p.M) was irradiated in the absence (30 Gy) or presence (55 Gy) of OH" scavengers leading to a residual lysozymeactivityof either 100%, or 75%. After SDS-PAGE, Comassie blue staining and scanning of resulting protein bands, results are expressed as % of total bands. Each value represents the mean + S.E. of n different experiments: * P < 0.05; ** P < 0.02; *** P < 0.001, treated versus tysozymealone. Fragments and aggregatesof lysozymein % of total lysozyme Irradiation dose (Gy) Conditions leading to 100% residual activity 6.9/~M lysozyme(n = 8) +34 ~.M uric acid (n = 3) + 34 ~M Ptx (n = 3) + 47/zM thymine(n = 4)

0 55 55 55

Conditions leading to 75% residual activity 6.9/J.M lysozyme(n = 8) +0.72/zM uric acid (n = 3) +5.1 ~M Ptx(n = 3) + 17.2 izM thymine (n = 4)

30 55 55 55

6.5-8 kDa

0 0 0 0 15_+4 21+5 345:1 * 1+1"

14.4 kDa

28 kDa

42 kDa

100 88_+ 7 ** 88+ 3 ** 44-+10 ***

0 0 0 44-+14 **

0 0 0 10_+5 **

66_+ 4

23_+ 3

2_+1

49_+ 3"* 46_+ 4 ** 58-+ 9

30+ 6 20_+ 4 32+11

0 0 8-+3"

15 TABLE II Scavenger effects on OH'-induced alteration of the fluorescence of lysozyme tryptophan and bityrosine

Lysozyme (6.9 tzM) was irradiated at 55 Gy in the presence of various concentrations of uric acid, Ptx and thymine. Tryptophan (ex, 290 nm; em, 360 nm in 6 M guanidine) is expressed as a percentage of initial (non-irradiated) lysozymefluorescence. Bityrosine (ex, 315 nm; em, 410 nm in 6 M urea) is expressed as nM of bityrosine formed (calibration curve as described in Materials and Methods).

Non-irradiated lysozyme Irradiated lysozyme + Thymine 3.4/~M 6.8/xM 17.2 jzM 47.0/xM

Tryptophan fluorescence (%) 100 56

Bityrosine formation (nM) 0 54

78 83 74 80

1 9 12 11

+ Uric acid 0.72/zM 1.72/zM 6.8 tzM 17.2/zM 34.0 ]zM

81 73 81 87 83

10 4 4 4 3

+ Pentoxifylline 3.4/xM 5.1/xM 6.8 ~M 34.0/zM

78 79 85 89

4 5 8 6

case, 59% of the enzyme activity was present in fraction 32, and 11% in fractions 13 and 26. The thymine radical thus protected the activity of lysozyme but not its structure, whereas uric acid and Ptx protected both. Discussion We confirm here that the hydroxyl radical O H ' alters lysozyme structure and activity and show that different O H ' scavengers have different effects. Lysozyme was attacked by OH" with a yield of 6.5 mol O H "/mol lysozyme, whereas Aldrich and Cundall [11] found a yield of 2.6 mol. This difference may reside, however, in differences in the egg lysozyme used. The O H - i n d u c e d decrease in enzyme activity correlated with the disappearance of the protein band (14.4 kDa) and the loss of tryptophan fluorescence. Aggregates (28 and 42 kDa) appeared above a dose of 20 Gy and their amount was correlated to the increase in bityrosine fluorescence, suggesting that they result from bityrosine f o r m a t i o n b e t w e e n lysozyme m o l e c u l e s [14,35,36]. This result differs from that of Aldrich and Cundall [11] who found only trimers and native lysozyme, but it is possible that SDS-PAGE is more sensitive for detecting these products than the gel filtration technique they used. O H induced about 10%

of specific fragmentation (6.5 and 8 kDa), a figure similar to that found for lactic dehydrogenase, transferfin and hexokinase [6]. Increasing concentrations of O H scavengers (Ptx, thymine and uric acid) gave increasing protection of lysozyme activity, showing that none of the scavengers or their secondary free radicals were toxic. The protection of the structure of lysozyme, however, differed according to the scavenger used. Uric acid and Ptx completely preserved secondary structure, and almost completely prevented the decrease in tryptophan fluorescence and bityrosine formation. In contrast, thymine increased bityrosine fluorescence, and aggregates retained lysozyme activity. As these aggregates were still active, the thymine radical formed by OH" would appear to protect tryptophan but not tyrosine; uric acid and Ptx radicals, on the other hand, appear to protect both tryptophan and tyrosine. To assess whether the concentrations of scavengers protecting lysozyme against O H ' attack were in agreement with a simple rate constant-dependent equation, we calculated first the distribution of OH" between lysozyme and the scavengers and second the theoretical yield (G t) and real yield (G r) of lysozyme attack. The distribution of O H between lysozyme and the scavengers was calculated by the competition ratio k S • C s / k L • CL, where k S is the scavenger rate constant, C S the scavenger concentration, k L the lysozyme rate constant and C L the lysozyme concentration [37]. For a ratio equal to 1, the hydroxyl radicals distribute equally between the lysozyme and scavenger. In the case of uric acid, which protected 100% of lysozyme activity at a concentration of 7 / z M , this ratio was 0.37 (73% O H on lysozyme and 27% on uric acid); Ptx protected 100% of lysozyme activity at 7/xM and had a ratio of 0.39 (72% OH" on lysozyme and 28% on Ptx). Thymine gave 100% protection at 25 /zM and had a ratio of 0.90 (53% OH" on lysozyme and 47% on thymine). The theoretical (G t) and real yield (G r) of lysozyme attack were also calculated as the amount of inactivated lysozyme at various scavenger concentrations irradiated with a dose of 55 Gy and expressed as /zM of lysozyme inactivated per Gy. The ratio ( G t ) / ( G r) was plotted as a function of log C s . k S (Fig. 6). If simple homogenous competition occurred, the ratio G t / G r would be equal to 1 [38]. In fact, it increased as a function of log C s -k s for the three scavengers, showing that non-homogeneous competition reactions occurred in every case. These values clearly show that the protection observed experimentally is much greater than that expected by these calculations. One hypothesis to explain the protective effects of scavengers depends on the properties of their radicals produced with O H ". As the oxidation of lysozyme by OH" gives rise to tryptophan and tyrosine radicals, they would react with reducing radicals. It is known that

16 scavengers for hydroxyl radicals play an important role in the scavenging capacity of the molecules studied.

5

0

Acknowledgements

3'

We are very grateful to Prof. Ferradini, Facult6 Paris V (rue des Saints P~res), and to Prof. Averbeck at the Institut Pierre et Marie Curie who allowed us to perform radiolysis experiments in their laboratories.

2' 1 "// 0

, 3

, 4

, 5

l o g C s x ks (s-,)

Fig. 6. Ratios of theoretical yield/real yield (Gt/G,) for OH" lysozyme attack in the presence of scavengers. G t / G r were calculated for 6.9/.~M lysozyme irradiated at the different concentrations of scavengers used in Table II. G t / G r is plotted against the log scavenging capacity per second (s-l). Each point represents the mean of four experiments ((e), Ptx; ( • ), thymine; ( t3 ), uric acid).

hydroxyl attack of uric acid gives 80% of reducing radical forms and 20% of oxidant radical forms, while thymine gives 56% of reducing radical forms [25,26]; we have previously shown that Ptx gives 66% of reducing radical [23]. The protection of lysozyme by these scavengers could correlate to the amount of reducing radicals, since uric acid had the lowest concentration giving 50% protection and gave the largest amount of reducing radical. However, protection by reducing radicals also depends on the rate of electron donation. This needs to be established by further studies to validate our hypothesis. With this restriction in mind, we thus propose a scheme of reactions between lysozyme and the different scavengers, designated as L and S, respectively. kL

L+OH'-}L' ks

(1)

S + O H ' ~ S o x +S "l~

(2)

Six + L ' - , S,~x + LL

(3)

S~ed + L ' ~ SredL "¢ S + L

(4)

In reaction 3, the scavenger radical would lead to the formation of aggregates (case of thymine). In reaction 4, the reducing radical would repair lysozyme radicals by binding to lysozyme (case of uric acid and Ptx). It can be assumed that the reducing radical repairs the enzyme by binding to the oxidized amino acid (e.g., Trp-108) or by binding to tyrosine and thus preventing bityrosine aggregate formation. The protection of lysozyme attack, by O H scavengers seems to be related to the nature of the secondary free radicals formed as they are able to inhibit tyrosyl radical binding and tryptophan oxidation; moreover, the competition kinetics between lysozyme and

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