Fast repair of protein radicals by urate

Free Radical Biology & Medicine 52 (2012) 1929–1936

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Original Contribution

Fast repair of protein radicals by urate Anastasia S. Domazou ⁎, Hongping Zhu, Willem H. Koppenol Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich CH-8093, Switzerland

a r t i c l e

i n f o

Article history: Received 15 November 2011 Revised 17 February 2012 Accepted 28 February 2012 Available online 8 March 2012 Keywords: Protein oxidation Protein repair Protein radical Antioxidant Urate Urate radical Rate constant Tryptophan radical Tyrosine radical Pulse radiolysis Free radicals

a b s t r a c t The repair of tryptophan and tyrosine radicals in proteins by urate was studied by pulse radiolysis. In chymotrypsin, urate repairs tryptophan radicals efficiently with a rate constant of 2.7 × 108 M− 1 s− 1, ca. 14 times higher than the rate constant derived for N-acetyltryptophan amide, 1.9 × 10 7 M− 1 s− 1. In contrast, no repair of tryptophan radicals was observed in pepsin, which indicates a rate constant smaller than 6 × 10 7 M− 1 s− 1. Urate repairs tyrosine radicals in pepsin with a rate constant of 3 × 10 8 M− 1 s− 1―ca. 12 times smaller than the rate constant reported for free tyrosine―but not in chymotrypsin, which implies an upper limit of 1 × 106 M− 1 s− 1 for the corresponding rate constant. Intra- and intermolecular electron transfer from tyrosine residues to tryptophan radicals is observed in both proteins, however, to different extents and with different rate constants. Urate inhibits electron transfer in chymotrypsin but not in pepsin. Our results suggest that urate repairs the first step on the long path to protein modification and prevents damage in vivo. It may prove to be a very important repair agent in tissue compartments where its concentration is higher than that of ascorbate. The product of such repair, the urate radical, can be reduced by ascorbate. Loss of ascorbate is then expected to be the net result, whereas urate is conserved. © 2012 Elsevier Inc. All rights reserved.

Oxidation of proteins, DNA, and cellular membranes by partly reduced oxygen species―radicals or molecules―(PROS; commonly designated as ROS) plays a key role in aging and various diseases [1,2]. Proteins are considered the major targets of PROS and, indeed, increased levels of protein oxidation products are measured under those conditions [3–5]. Precursors of these products are protein and protein peroxyl radicals (Pr• and PrOO •, correspondingly). When proteins are exposed to PROS, carbon-center radicals Pr• are expected to be initially formed, which are both oxidizing and reducing agents [6]. In living organisms, Pr• would then react further by various pathways that either propagate biological damage―such as reaction with DNA or lipids, conversion to other protein radicals (Pr′ •), reaction with oxygen (O2) to form oxidizing PrOO•―or alleviate damage―such as repair by antioxidants, which are part of the defense system [7]. Ascorbate (Hasc −), uric acid, and glutathione (GSH) are considered the most important endogenous antioxidants [8], whereas carotenoids and Abbreviations: Pr•, protein radical; PrOO•, protein peroxyl radical; H3ur, uric acid; H2ur−, urate [dihydrogenurate(1−)]; Hur•−, urate radical; Hasc−, ascorbate [monohydrogenascorbate(1−)]; asc•−, ascorbyl radical; GSH, glutathione; Trp and TrpH, tryptophan; Trp•, tryptophan radical; Tyr and TyrOH, tyrosine; TyrO•, tyrosine radical; HSA, human serum albumin; Lz, lysozyme; Lac, lactoglobulin; Chy, α-chymotrypsin; Pep, pepsin; N-ac-TrpH-NH2, N-acetyltryptophan amide; PrOOH, protein hydroperoxide; N3• , azide radical (trinitrogen(•)); CTAB, cetyltrimethylammonium bromide; SDS, sodium dodecyl sulfate. ⁎ Corresponding author. Fax: + 41 446321090. E-mail address: [email protected] (A.S. Domazou). 0891-5849/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2012.02.045

flavonoids, introduced by dietary uptake, are among the important exogenous ones [8]. Their antioxidant activity is commonly thought to be based on their ability to scavenge HO• in vivo. However, this would require very high concentrations of such antioxidants. Instead, aside from other processes, these antioxidants can function also by repair, as shown here for urate and elsewhere for ascorbate [9,10]. Uric acid, an end product of human purine metabolism [11], is present at high concentration in plasma and in saliva (about 0.3 and 0.15 mM, respectively) [12–14], where it contributes to approximately 50 and 70%, respectively, of the antioxidant capacity [13,15–17]. It has been proposed that the loss of uricase in humans―responsible for the high levels of uric acid―is related to the loss of their ability to synthesize ascorbic acid [18] and that both might be associated with dietary changes during the evolution [19]. There is evidence that uric acid levels are related to the longevity of humans [20–22]. Decreased levels are measured in patients that suffer from multiple sclerosis [23–25]. Its physiological role as an antioxidant is, presumably, to scavenge radicals [26] and to chelate transition metals [27]. At physiological pH, uric acid is present in its monoanion form, H2ur − (pKa1 = 5.4 and pKa2 = 9.8 [28]). The one-electron oxidation product of H2ur − is the urate radical Hur •− (pKa1 = 3.1, pKa2 = 9.5) and the electrode potential at pH 7 is E°′(Hur •−, H +/H2ur −) ~ + 0.59 V (relative to the normal hydrogen electrode, NHE) [28] (Fig. 1). This value is lower than those of amino acid radicals and makes a reaction of H2ur − with these radicals thermodynamically possible. Mechanistic studies indicated that, in addition to its ability to react with hydroxyl

1930

A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936

E°´ (V)

1.2

Germany). All chemicals were of the highest quality available and were used as received. Water was purified in a Millipore Milli-Q unit.

a b

0.8

d

c 0.4

Pulse radiolysis

e

0.0 0

5

10

15

pH Fig. 1. pH dependence of the electrode potential (E°′) of the urate radical/urate couple. Open circles: experimentally measured E°′ values [28,66]. Black line: calculated electrode potential E°′ of the couples (a) H2ur•, H+/H3ur; (b) Hur•−, 2H+/H3ur; (c) Hur•−, H+/H2ur−; (d) U•−, 2H+/H2ur−; (e) U•−, H+/Hur2− by use of the Nernst equation and the measured E°′ value at pH 8.9, E°′(Hur•−, H+/H2ur−) = + 0.47 (vs NHE) [28]. Black squares: calculated E°′ values at pH 0, 3.1, 5.4, 9.5, 9.8, and 13.

and peroxyl radicals, and with peroxynitrite [28–32], H2ur − can repair or prevent oxidative damage in biomolecules [12,33–35]. For instance, H2ur − repairs nucleobase and DNA radicals [28,33,34], prevents lipid and protein oxidation in bovine milk induced by H2O2 activation of endogenous peroxidases or light [35], and inhibits the oxidation of the low-density lipoprotein (LDL) stimulated by Cu 2+ [12,36]. LDL oxidation has been proposed to be triggered by oxidation of one or several 37-Trp residues in apolipoprotein B, the protein moiety of LDL [37]. Although, in general, the antioxidant properties of H2ur − have received extensive attention, only a few studies have so far dealt with the repair of protein radicals by H2ur − [38,39]. It has been reported that H2ur − reacts with bovine serum albumin radicals, in which the unpaired electron is thought to be on a tyrosine [40], and the Trp-14 peroxyl radical of myoglobin [39]. Moreover, H2ur − repairs tryptophan radicals (Trp •), free and bound in human serum albumin (HSA) or lysozyme (Lz), with rate constants that vary from 10 7 to 10 8 M − 1 s − 1 [10,30,38], and free tyrosine radicals (TyrO •) in CTAB micelles [38] and Lz(TyrO •) in SDS micelles [10] with rate constants of 3.5 × 10 9 and 5.4 × 10 6 M − 1 s − 1, respectively. Recently, we reported on the repair of protein radicals by the other two antioxidants, Hasc − and GSH [9,41]. The rate constants found for the reaction of Hasc − with TyrO • and Trp • are on the order of 10 7 and 10 8 M − 1 s − 1, respectively. Whereas Hasc − reacts so fast with Pr • that formation of PrOO • is possibly prevented, GSH reacts substantially slower: rate constants of GSH reactions with TyrO • and free and lysozyme-bound Trp • range from 10 3 to 10 5 M − 1 s − 1 [41]. It is of importance to elucidate whether H2ur − is able to repair protein radicals and, moreover, whether such a process takes place at rates that are beneficial. This depends on the probabilities of reactions of the protein radicals with H2ur −, Hasc −, and GSH, and this, in turn, on the corresponding rate constants and concentrations of the three antioxidants. Therefore, we studied, by pulse radiolysis, the reaction of H2ur − with radicals generated on α-chymotrypsin (Chy), pepsin (Pep), and N-acetyltryptophan amide (N-Ac-TrpH-NH2). We found that the ability of H2ur − to inhibit electron transfer and to repair Trp • and TyrO • in proteins strongly depends on the protein. Our results, together with those reported by others for human serum albumin and lysozyme, suggest that H2ur −, like Hasc −, repairs protein radicals and prevents damage in vivo. Experimental procedures Materials α-Chymotrypsin from bovine pancreas, NaH2PO4, Na2HPO4, and NaN3 were purchased from Fluka (Buchs, Switzerland). N-acetyltryptophan amide was from Bachem (Bubendorf, Switzerland). Pepsin from porcine gastric mucosa and uric acid were supplied by Sigma–Aldrich (Steinheim,

Electron pulses of 2.0 MeV and 50-ns duration were generated by a Febetron 705 accelerator (Titan Systems Corp., San Leandro, CA, USA). The light source was a 75-W xenon arc lamp (Hamamatsu, Schüpfen, Switzerland) and the optical path length of the quartz cell (Hellma GmbH & Co KG, Zumikon, Switzerland) was 1 cm. Before each pulse, a fresh sample of 300 μl volume was introduced into the cell. The detection system consisted of a Roper Scientific Acton SP300i monochromator (Ottobrunn, Germany) and a Hamamatsu R928 photomultiplier (Schüpfen, Switzerland), a Femto Messtechnik GmbH DHPCA-200 or HCA-100 M-50 k-C amplifier (Berlin, Germany), and a Yokogawa Electric Corp. DL7100 digital storage oscilloscope (Tokyo, Japan). The dose per pulse used was 7–25 Gy and was determined by thiocyanate dosimetry [42]. Results were analyzed with Kaleidagraph from Synergy Software (Reading, PA, USA) or simulated with the gen-int5 simulator (EXCEL-Macro). Error bars in the figures represent standard deviations (s) from the mean value. Secondorder rate constants are given as mean value± tss/n 0.5; second-order rate constants derived from linear plots of kobs versus protein concentration are given as mean values ± tss) for 95% probability. Generation of amino acid and protein radicals N-Ac-Trp •-NH2 or protein radicals were generated by irradiating N-Ac-TrpH-NH2 or protein solutions in 5 mM phosphate buffer, pH 7.4, that contained 0.1 M NaN3, 0–500 μM H2ur − and were saturated − with N2O (24 mM) at room temperature. Hence, practically all eaq are converted to HO • [43] and these, in turn, quantitatively to N3• [29]. N3• selectively oxidizes Trp and Tyr residues in the protein with formation of the corresponding Trp • and TyrO • [9,10,44–46] within a few microseconds. At the millimolar protein concentration used here, − about (i) 3–5% of the HO • and (ii) 10–15% of eaq is expected to react directly with the protein and not with the N3− and N2O, respectively. Thus, radicals located on the amino acids Trp and Tyr, but also on cystine and histidine, could be formed, with maximal total yield of about 10%. Similarly, ≤5% of HO • is expected to react directly with N-AcTrpH-NH2. N-Ac-TrpH-NH2 was studied as model of the Trp residues in peptides and proteins. Kinetics of reactions of amino acid and protein radicals Formation and decay of N-Ac-Trp •-NH2, protein radicals, and Hur •− were followed by measurement of transient absorbance changes at 340, 405, and 510 nm. We assumed that the molar absorption coefficients of Trp • and TyrO • in proteins and free in solution are identical [44,47]. At 510 and 405 nm, the ε values were taken from the literature, whereas those at 340 nm were deduced from the published absorbance spectra [46,48,49]. From the two ε values deduced at 340 nm for Trp •, the higher one is quite close to the one we derive for Trp • in the proteins from our absorbance measurements, by using the ε values given in Table 1 for both radicals at 510 and 405 nm and that for TyrO • at 340 nm. However, for N-Ac-Trp •-NH2 we calculated, based on a 100% conversion of N3• to N-Ac-Trp •-NH2 and bandwidth of 5 nm, ε = 2000 M − 1 cm − 1 at both 340 and 510 nm, because the absorbance measured at those wavelengths was identical. The ε values for Hur •− were derived from separate experiments in the absence of proteins and, considering differences in the experimental conditions, such as bandwidth, are in agreement with those deduced from its known absorbance spectrum [28,30]. We used these ε values (Table 1) to calculate radical yields and to perform kinetic simulations.

A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936 •

510 nm a

N-Ac-Trp -NH2 Pr(Trp•) Pr(TyrO•) Hur•− a b c d

2000 1800b 70b 500d

•

405 nm

•

340 nm

a

300 300b 2600b 4000d

2000 1800c,i, 2400c,ii 600c,iii 6500d, 6000a

• • N
Ac
TrpH
NH2 þ N3 →N
Ac
Trp
NH2 − • •− − þ H2 ur þ N3 →Hur þ N3 þ H

þ

− N3

þH

þ

•

N
Ac
Trp
NH2 þ H2 ur →N
Ac
TrpH
NH2 þ Hur þ Hur •

•−

→products •−

N
Ac
Trp
NH2 þ Hur →products

ð1Þ ð2Þ

2N
Ac
Trp
NH2 →products

ð3Þ •−

þ

ð8Þ

•

ð9Þ

•

ChyðTrp Þ→products

ð10Þ

•

ð11Þ

•

ð12Þ

ChyðTyrO Þa →products ðfastÞ

•

We used Reactions (1)–(6) for the simulations of reactions of N-Ac-Trp-NH2 and Reactions (2), (5), and (7)–(14) for chymotrypsin. Reaction (9) represents both intra- and intermolecular electron transfer processes―shown as Reactions (9i) and (9ii) under Results― under constant and in large excess concentration of chymotrypsin. Under these conditions, electron transfer would obey pseudo-firstorder kinetics with k9 = k9i + k9ii[Chy]. Chy(TyrO •)a represents only TyrO • generated directly by reaction with N3• (located on one or more Tyr residues), whereas Chy(TyrO •)b represents only TyrO • generated by intra- or intermolecular electron transfer (located on one or more Tyr residues).

•−

−

−

ChyðTrp Þ þ H2 ur →ChyðTrpHÞ þ Hur

Kinetic simulations

Hur

•

ð7Þ

ChyðTyrO Þb →products ðslowÞ

The rate constants of the reactions of Trp • in N-Ac-TrpH-NH2 and chymotrypsin with H2ur − were obtained from the initial part of the absorbance transients at λ ≥ 500 nm, where interference from the decay of Hur •− is negligible; below 500 nm, the absorbance of Hur •− is too strong. The rate constant of the reaction of Pep(TyrO •) with H2ur − was calculated from the rates of formation of Hur •−, which were obtained from the difference between transient absorbance changes at 340 nm in the presence and the absence of H2ur −. Absorbance transients due to only Pep(TyrO •) were also calculated: at 340 nm the concentration of Hur •− was determined, as described above. This allows us to calculate its contribution to the absorbance at 405 nm. Similarly, from the transient absorbance at 510 nm, due to Pep(Trp •), the absorbance contribution of Pep(Trp •) at 405 nm was calculated. These two corrections make it possible to determine the absorbance change at 405 nm due to Pep(TyrO •). To avoid N3• –N3• recombination (k = (3–4.5) × 10 9 M − 1 s − 1 [46,50]) the radiation dose was kept low, [N3• ] was b13 μM, and the proteins and N-Ac-TrpH-NH2 were present in large excess (2 or 5 mM N-Ac-TrpH-NH2, 0.2 or 2 mM chymotrypsin, 3 mM pepsin). For N-Ac-TrpH-NH2, to study the reaction between H2ur − and Trp •, very low doses of 4–7 Gy were used, to minimize the interference of the Trp •–Trp • recombination. To ensure pseudo-first-order conditions, the concentration of H2ur − was kept in at least sixfold excess over that of N-Ac-Trp •-NH2 or protein radicals. Under these conditions, the extent of reaction of N3• with H2ur − (k2 = 5.4 × 10 9 M − 1 s − 1 [10]) was also minimized. Because of the high concentration of the protein, the viscosity increases macroscopically. However, in the space between the protein molecules, the viscosity of the solution does not change significantly.

−

þ

ChyðTrp Þ→ChyðTyrO Þb

a

This work. Refs. [44,47]. Deduced from (i) Fig. 1 in [49], (ii) Fig. 5 in [48], and (iii) Fig. 2 in [46]. Deduced from Fig. 1 in [30] and Fig. 3 in [28].

•

−

ChyðTyrOHÞ þ N3 →ChyðTyrO Þa þ N3 þ H

ε (M− 1 cm− 1)

•

•

ChyðTrpHÞ þ N3 →ChyðTrp Þ þ N3 þ H

Table 1 Molar absorption coefficients of amino acid, protein, and urate radicals. Radical

1931

ð4Þ ð5Þ ð6Þ

•

−

•−

ChyðTyrO Þa;b þ H2 ur →ChyðTyrOHÞ þ Hur

ð13Þ •−

ð14Þ

For k1, k2, k7, and k8 literature values were used [9,10] and for k3, k5, k9–k12, and k14 those measured in this work. The values for k4 and k13 were allowed to vary. Results N-acetyltryptophan amide In the absence of H2ur −, N-Ac-Trp •-NH2, formed in Reaction (1), decays by second-order kinetics because of radical–radical recombination, Reaction (3) (Figs. 2A and B, trace a), with a rate constant (Table 2) identical to that reported previously [9] and quite close to that reported for free Trp • [51]. In the presence of H2ur −, two consecutive processes are observed (Figs. 2A and B, trace b). The fast one―characterized by an accelerated absorbance decay at 510 nm and a concurrent absorbance growth at 340 nm―corresponds to repair of N-Ac-Trp •-NH2 by H2ur − accompanied by formation of Hur •−, Reaction (4). The slower process―characterized by absorbance decay at both wavelengths―corresponds to the second-order decay of Hur •−, Reaction (5); the rate constant (Table 3) agrees with the literature [28,30]. When [H2ur −] > 200 μM, the fast decay fits a single exponential with a rate constant, kobs, that is linearly dependent on the H2ur − concentration (Fig. 2C), from which a second-order rate constant k4 was derived (Table 3). α-Chymotrypsin Chymotrypsin contains 8 Trp (positions 27, 29, 51, 141, 172, 207, 215, and 237), 4 Tyr (positions 94, 146, 171, and 228), and 10 Cys, all involved in disulfide bonds (Cys1–Cys122, Cys42–Cys58, Cys136– Cys201, Cys168–Cys182, Cys191–Cys220). The experiments with chymotrypsin and N3• in the absence of H2ur − confirm in general our previous results [9]: both Chy(Trp •) and Chy(TyrO •) are formed in the ratio 5:1, Reactions (7) and (8); Chy(Trp •) decays partly by electron transfer from TyrOH to Trp •, Reactions (9i) and (9ii) (Fig. 3) [9,52], and the remaining Chy(Trp •) disappears by other pathways, Reaction (10): •

•

ChyðTrp ; TyrOHÞ→ChyðTrpH; TyrO Þ •

ð9iÞ •

ChyðTrp Þ þ ChyðTyrOHÞ→ChyðTrpHÞ þ ChyðTyrO Þ

ð9iiÞ

In addition to our earlier observations, the decay of Chy(Trp •) shows a dependence on the protein concentration; it can be fitted to a single exponential at high protein concentrations. At low protein concentrations, the decay of Chy(Trp •) shows a fast and a slow part and can be fitted by two single-exponentials (Figs. 3A and B, Table 2), indicating that at least two different Chy(Trp •) moieties are formed. The fast decay is accompanied by formation of Chy(TyrO •) at the same rate as the disappearance of Chy(Trp •); formation of

1932

A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936

A

Table 3 Rate constants of reactions of H2ur− with amino acid and protein radicals.

8 6

Amino acid/protein radical

4

•

b

0 0

0.4

0.8

1.2

Time (ms)

B 15

b

10

5

a

0 0

1

2

3

Time (ms)

C12 8

4

0 0

200

400

600

Fig. 2. N-Ac-Trp•-NH2 and its reaction with H2ur−. N2O-saturated aqueous solutions of 5 mM N-Ac-TrpH-NH2 containing 0.1 M NaN3, 5 mM phosphate buffer, and various amounts of H2ur− were irradiated: dose 5–7 Gy, pH 7.4, room temperature. (A) Absorbance decay at 510 nm in the absence (trace a) and presence (trace b) of 500 μM H2ur−. (B) Absorbance changes at 340 nm in the absence (trace a) and presence (trace b) of 400 μM H2ur−. (C) kobs for the decay of N-Ac-Trp•-NH2 as a function of H2ur− concentration. The smooth lines superimposed on the traces in (A) and (B) represent the fit of the simulation. The kobs values in (C) are derived from the single-exponential fit to the 510nm traces.

Chy(TyrO •) is not observed during the slow decay, however, such a formation would have been masked by the decay of Chy(TyrO •) formed during the fast process. Moreover, the yield of conversion of

Trp• decay (k (s− 1))

N-Ac-TrpH-NH2a Chymotrypsin 0.2 mMa 1 mMc 2 mMa Pepsin 1 mMc 3 mMa,e

b

a

Transfer Trp• → Tyr (%)

7 × 103, ~ 500 8 × 103 9 × 103

nd nd 103, ~ 30

20 40

3.1 × 104 8.8 × 104

500, ~ 50 2 × 103, ~ 150

94 98

d

c

a

b 1 × 106 a

3 × 108

For the Hur•−–Hur•− recombination, we measured 2 k5 = (2.4–3.5) × 108 M− 1 s− 1 dependent on the conditions. a Values derived from kinetic simulations.

Chy(Trp •) to Chy(TyrO •) increases with increasing protein concentration (Fig. 3, traces a and b; Table 2). These results imply that interand intramolecular electron transfers occur in parallel; at most only 10% of Chy(Trp •) is converted to Chy(TyrO •) intramolecularly. The decay of Chy(TyrO •) fits two single-exponentials (Table 2), which indicates the existence of at least two different Chy(TyrO •) moieties. Formation and decay of Chy(TyrO •) by multiple pathways results in its formation to peak slightly ahead of the complete decay of Chy(Trp •) (Fig. 3). In the presence of H2ur −, Chy(Trp •) decays faster, accompanied by formation of Hur •− (Figs. 4A and B). The absorbance in the range 340–600 nm, recorded after the complete decay of Chy(Trp •) (Fig. 4C), corresponds nearly completely to the spectrum of Hur •− [28,30] with a small contribution from Chy(TyrO •), being present at low concentrations; below 340 nm the products of Reaction (5) also absorb. These data are consistent with repair of Chy(Trp •), Reaction (13), and, thereby, inhibition of electron transfer by H2ur −. Hur •− forms with a yield of maximum 80% relative to Chy(Trp •) and 70% relative to all protein radicals and decays according to

A

20 15

b

10

a

5 0 0

1000

2000

3000

Time (µs)

B 10 a

6 TyrO• decay (k (s− 1))

4

b

0

This work. N-Ac-Trp•-NH2 decayed with 2 k = (7.7 ± 0.9) × 108 M− 1 s− 1. Ref. [9]. d Based on these values, the rate constants for intra- and intermolecular electron transfer are estimated: 2.5 × 103 s− 1, 3 × 107 M− 1 s− 1. e Late formation of Trp• with k = 400 s− 1. b

(1.9 ± 0.4) × 10 (1.8 ± 0.2) × 107 (2.7 ± 0.5) × 108 (2.8 ± 0.2) × 108 b 6 × 107

2

d

TyrO• 7

8

Table 2 Decay of amino acid and protein radicals. Amino acid/ protein

Trp•

N-Ac-Trp -NH2 N-Ac-Trp•-NH2 Chymotrypsin(Trp•, TyrO•) Chymotrypsin(Trp•, TyrO•) Pepsin(Trp•, TyrO•)

a

2

k (M− 1 s− 1)

0

200

400

600

Time (µs) Fig. 3. Electron transfer from Tyr to Trp• in chymotrypsin and its dependence on protein concentration. N2O-saturated aqueous solutions of chymotrypsin containing 0.1 M NaN3 and 5 mM phosphate were irradiated; pH 7.4, room temperature. (A) Decay of Chy(Trp•) in 2 (trace a) and 0.2 mM (trace b) chymotrypsin; dose 16 Gy, detection at 510 nm. (B) Formation of Chy(TyrO•) in 2 (trace a) and 0.2 mM (trace b) chymotrypsin; dose 8 Gy, detection at 405 nm. The smooth lines superimposed on the traces in (A) and (B) represent the fit with a single exponential (traces a) or two single exponentials (traces b).

B Absorbance (×10 )

3

8 6

a

4 2

b 0 0

b

800

30 20 10 0 0

1200

Time (µs)

D

3

a 100

200

Time (µs) 4

3

Absorbance (×10 )

C

400

1933

40

3

A 10 Absorbance (×10 )

A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936

3

2

2 1

1 0

0 300

400

500

600

0

40

80

120

Wavelength (nm) Fig. 4. Chymotrypsin radicals and their reactions with H2ur−. N2O-saturated aqueous solutions of 2 mM chymotrypsin containing 0.1 M NaN3, 5 mM phosphate, and various amounts of H2ur− were irradiated: pH 7.4, room temperature. (A) Absorbance decay at 510 nm in the absence (trace a) and presence (trace b) of 80 μM H2ur−: dose 10 Gy. (B) Absorbance changes at 340 nm in the absence (trace a) and presence (trace b) of 80 μM H2ur−: dose 16 Gy. (C) Absorbance spectrum 80 μs after the pulse (dose 25 Gy) in the presence of 100 μM H2ur−: normalized dose 1 Gy. (D) kobs for the decay of Chy(Trp•) as a function of H2ur− concentration. The smooth lines superimposed on the traces in (A) and (B) represent the fit of the simulation. The kobs values in (D) are derived from the single-exponential fit to the 510 nm traces.

Reaction (5). The decay of Chy(Trp •) fits to a single exponential with rate constants linearly dependent on H2ur − concentration (Fig. 4D), from which the second-order rate constant k13 was derived (Table 3). Observation of a reaction between H2ur − and Chy(TyrO •) would be partially prevented by the reaction of H2ur − with Chy(Trp •) and the fast reaction of Hur •− with itself, Reactions (13) and (5), respectively. However, the relative absorbance changes with time at 340 and 405 nm, after correction for Chy(Trp •) decay, are proportional to the corresponding ε values of Hur •−. This―given the very slow decay of Chy(TyrO •) in the absence of urate―indicates that a reaction between H2ur − and Chy(TyrO •) does not occur under our experimental conditions. Indeed, such reaction would have resulted in lower absorbance increase at 405 nm, because not only formation of Hur •−, but also parallel decay of Chy(TyrO •), would have occurred. This observation, combined with the rate constant for the fast decay of Chy(TyrO •), sets an upper limit for the rate constant of the reaction of H2ur − with TyrO • of 10 6 M − 1 s − 1 (Table 3).

Pepsin Pepsin has 5 Trp (positions 39, 141, 181, 190, and 299), 16 Tyr (positions 9, 14, 44, 75, 86, 113, 114, 125, 154, 174, 175, 189, 267, 274, 309, 310), and 6 Cys, all involved in disulfide bonds (Cys45– Cys50, Cys206–Cys210, Cys249–Cys282). As in the case of chymotrypsin, our experiments with pepsin in the absence of H2ur − confirm the reported initial formation of Pep(Trp •) and Pep(TyrO •), Reactions (15) and (16), the electron transfer from Pep(TyrOH) to Pep(Trp •) [9,52,53], and the late formation of some Pep(Trp •) [9]. •

•

−

PepðTrpHÞ þ N3 →PepðTrp Þ þ N3 þ H •

•

−

þ

ð15Þ þ

PepðTyrOHÞ þ N3 →PepðTyrO Þ þ N3 þ H

ð16Þ

Decay of both radicals is dependent on the protein concentration: at a higher protein concentration, higher rate constants were measured (a) for the first-order conversion of Pep(Trp •) to Pep(TyrO •) by electron transfer, which is consistent with both intra- and

intermolecular electron transfer [52], with kET = kET,intra + kET,inter [Pep], and (b) for the decay of Pep(TyrO •), which fits two single exponentials (Table 2 [9,52]), indicating that at least two different Pep(TyrO •) moieties are formed. H2ur − does not affect the decay of Pep(Trp •) (Fig. 5A). This indicates that the reaction of H2ur − with Trp • cannot compete with the fast electron transfer from Tyr to Trp •, which together with kET sets an upper limit for the rate constant for the reaction between H2ur − and Trp • of 6 × 10 7 M − 1 s − 1 (Table 3). However, the absorbance changes at 340 nm (Fig. 5B) and the acceleration of the decay observed at 405 nm (Fig. 5C) are consistent with formation of Hur •−, which is partially masked by the simultaneous decay of Pep(Trp •) and Hur •− via electron transfer and Reaction (5). This suggests that the following reaction takes place: •

−

PepðTyrO Þ þ H2 ur →PepðTyrOHÞ þ Hur

•−

ð17Þ

To gain insight into the reaction scheme and the kinetics, we corrected the absorbance changes at 340 and 405 nm (see Experimental procedures). The absorbance changes at 340 nm were corrected for contributions by Pep(Trp •) and Pep(TyrO •). These corrected absorptions are due nearly only to Hur •− (Fig. 6A), because H2ur − influences neither the formation and decay of Pep(Trp •) nor the formation of Pep(TyrO •), but only the decay of Pep(TyrO •). The yield of Hur •− measured from these corrected absorptions is maximal 46% relative to the initially formed Pep(TyrO •) by reaction with N3• , Reaction (16), and 20% relative to the total amount of Pep(TyrO •) formed by reaction with N3• and by electron transfer. The formation of Hur •− fits a single exponential (Fig. 6A) with rate constant linearly dependent on H2ur − concentration; k17 is listed in Table 3. Absorbance changes at 405 nm were corrected for contributions by Hur •− and Pep(Trp •). These corrected absorptions are due mostly to Pep(TyrO •) (Fig. 6B). In the presence of H2ur −, we observe a smaller increase in absorbance (Fig. 6B), although the formation of Pep(TyrO •) via Reaction (16) and electron transfer is not dependent on H2ur −. This indicates that a fraction of Pep(TyrO •) is scavenged by H2ur − within the first 100 μs, Reaction (17), which is consistent

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A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936

A

A 20

15

15 10 10 5

b a

0 0

B

5

50

0 100

150

0

200

20

d c b a

10

C

100

120

150

a

20

10

0 50

80

B 30

30

0

40

Time (µs)

Time (µs)

b

0

200

0

Time (µs)

50

100

150

200

Time (µs)

30

Fig. 6. Pep(TyrO•) and its reaction with H2ur−. N2O-saturated aqueous solutions of 3 mM pepsin containing 0.1 M NaN3, 5 mM phosphate were irradiated in the absence and presence of 150 μM H2ur−: dose 18 Gy, pH 7.4, room temperature. (A) Formation of Hur•− in the presence of 150 μM H2ur−. The trace is the difference between traces d and a of Fig. 5B. The smooth line superimposed on the trace represents the singleexponential fit. (B) Formation and decay of Pep(TyrO•) in the absence (trace a) and presence (trace b) of 150 μM H2ur−. Trace b was derived as described in the text.

a b c d

20

10

0 0

50

100

150

200

Time (µs) Fig. 5. Pepsin radicals and their reactions with H2ur−. N2O-saturated aqueous solutions of 3 mM pepsin containing 0.1 M NaN3, 5 mM phosphate, and various amounts of H2ur− were irradiated: dose 18 Gy, pH 7.4, room temperature. (A) Absorbance decay at 510 nm in the absence (trace a) and presence (trace b) of 150 μM H2ur−. (B) Absorbance changes at 340 nm in the absence (trace a) and presence of (trace b) 50, (trace c) 100, and (trace d) 150 μM H2ur−. (C) Absorbance changes at 405 nm in the absence (trace a) and presence of (trace b) 50, (trace c) 100, and (trace d) 150 μM H2ur−.

with k17. The subsequent decay fits a single exponential with rate constant independent of H2ur − concentration and identical to that measured for the fast decay of Pep(TyrO •) in the absence of H2ur −. Kinetic simulations The rate constants for the reaction of N-Ac-Trp •-NH2, Reaction (4), and of Chy(Trp •), Reaction (13), with H2ur − were also derived from the curves at 340 and 510 nm by means of kinetic simulations; these rate constants are practically identical to those measured at 510 nm (Table 3). The smooth lines superimposed on traces b in Figs. 4A and B represent simulations for chymotrypsin. For N-AcTrp •-NH2, the kinetic simulations at 340 nm fit to the experimental data (Fig. 2, traces b and smooth lines) only when the following reaction with k = (7.7 ± 0.8) × 10 8 M − 1 s − 1 is included: •

•−

N
Ac
Trp
NH2 þ Hur →unidentified products

ð6Þ

Discussion Pepsin and chymotrypsin contain both Tyr and Trp residues. Their reactions with N3• are quite similar, with formation of both Trp • and TyrO • [9,52], with the former as the preferred target. Trp • and TyrO • are formed at different ratios, depending on the protein, in agreement

with previous results. This ratio is, for each protein, independent of the protein concentration. In chymotrypsin, at least two different Trp • are formed. We observed the reported [9,52,53] electron transfer from Tyr to Trp • with TyrO • formation, which we found to be (a) dependent on the protein concentration und thus mainly intermolecular and (b) more pronounced and about an order of magnitude faster in pepsin: the yield was 10–40% in chymotrypsin and almost 100% in pepsin. Moreover, in both proteins, there are at least two different TyrO • moieties, formed by electron transfer and by N3• . Our finding that the decay of Trp • in pepsin is followed by a small amount of Trp • formation disagrees with the report that Trp • decays by a fast and a slow process [52]. This disagreement may be due to differences in experimental conditions, namely pH and protein concentration: [52] carried out their experiments at acidic pH and with pepsin concentrations that were 10 times lower. At pH >6, pepsin becomes irreversibly denatured [54] and more Tyr residues are exposed to the solvent at pH 7.4 [55]. Such a conformational change could alter the relative positions of Trp and Tyr residues and, thereby, influence the rate of electron transfer from Tyr to Trp •. The late formation of Trp • − could be related either to the direct reaction between eaq and pepsin or to electron transfer from Trp to TyrO •. Although such an electron transfer has not been observed at pH 7, it would be thermodynamically possible, because the electrode potentials E°′(Trp •, H +/TrpH) and E°′(TyrO •, H +/TyrOH) differ only by about 0.1 V and are dependent on the protein environment. Moreover, at lower protein concentrations, intermolecular electron transfer becomes less important. H2ur − repairs N-Ac-Trp •-NH2 as efficiently as free Trp • [10,30] (Fig. 7). In both pepsin and chymotrypsin, the efficiency of H2ur − to repair Trp • is largely dependent on the protein: the rate constant for repair of Chy(Trp •) and the upper limit for the rate constant for repair of Pep(Trp •) are about 14 and 4 times, respectively, higher than that for the free amino acid. Earlier studies showed that the repair of Trp • by H2ur − in HSA is about 10 times faster than repair of the free

A.S. Domazou et al. / Free Radical Biology & Medicine 52 (2012) 1929–1936

12

1935

protein-bound TyrO • and Trp • and H2ur − is proposed, which leads to repair of the protein and formation of Hur •−:

9

•

•

−

PrðTrp =TyrO Þ þ H2 ur →PrðTrpH=TyrOHÞ þ Hur

6

•

3

C

m

e

in Ly

so

zy

um

Al b

Se r

hy

um

m

ot

P

ry

ep

ps

si

n

in

2 H -N

pH

Ty

H

um

an

N

-A c-

Tr

Tr

pH

rO H

0

Fig. 7. Rate constants of the reaction of H2ur− with amino acid and protein radicals. Blue, Trp•; red, TyrO•; shaded columns, upper limit for the rate constant; (a) [10], (b) [30], (c) [38], (d) this work.

amino acid [38]; in contrast, the rate constant of repair of Trp • in lysozyme [10] is close to that of the free amino acid. These rate constants (Fig. 7) indicate that the insertion of Trp • in the protein does not constitute a barrier for the reaction with H2ur −. In many cases, the protein environment favors the repair process. There is no relation between protein size, which increases in the order Lz b Chy b Pep b HSA, and rate constants, because chymotrypsin, for which the rate constant of repair is highest, is not much larger than lysozyme, for which the rate constant is as high as for the free Trp •. In contrast, the repair of protein-bound Trp • by Hasc − is, in most cases, neither inhibited nor enhanced compared to free Trp •. Hasc − repairs protein-bound Trp • mainly with rate constants between 0.8 and 1.8 × 10 8 M − 1 s − 1, quite similar to free Trp • [9,10]. The only exception is the repair of Lac(Trp •), which is 5 times slower [9]. Taken together, H2ur − appears more efficient than Hasc − in repairing Trp • in chymotrypsin, whereas for lysozyme and pepsin Hasc − is better. The efficiency of H2ur − at repairing protein-bound TyrO • varies strongly with the protein, as shown by this study and other publications [10,38] (Fig. 7). In chymotrypsin, H2ur − appears unable to repair TyrO •, and the upper limit for the rate constant of repair of TyrO • is about 300 times lower than the corresponding rate constant in pepsin, 5 times lower than that measured by Hoey and Butler [10] in lysozyme in the presence of anionic SDS micelles, and 3600 times lower than that measured for free TyrO • in the presence of cationic CTAB micelles [19,38]. However, the presence of micelles affects the protein structure and also the rate constants. The ability of H2ur − to repair protein-bound TyrO • is very dependent on the protein, much more so than that of Hasc −. Indeed, in the case of Hasc −, the rate constants measured for chymotrypsin, pepsin, lysozyme, and insulin are on the order 10 7 M − 1 s − 1 and only somewhat lower than that for free N-ac-TyrO •-NH2 [9,10], which indicates the absence of a steric barrier. Taken together, H2ur − is more efficient than Hasc − in repairing TyrO • in pepsin, whereas Hasc − is superior in chymotrypsin and lysozyme. H2ur − inhibits electron transfer only in chymotrypsin and not in pepsin. Thus, in chymotrypsin, the fast reaction of H2ur − with Chy(Trp •) relative to Reactions (9) and (10) inhibits electron transfer from TyrOH to Trp •, whereas in pepsin, electron transfer is not inhibited, being much faster than the reaction of H2ur − with Pep(Trp •). It should be emphasized that, in the case of pepsin, electron transfer― being partly of intermolecular character―is dependent on protein concentration and thus, at a low enough pepsin concentration, H2ur − may inhibit electron transfer. Based on these results and those reported for human serum albumin [38] and lysozyme [10], the following general reaction between

•−

ð18Þ

•

The location and identity of Trp and TyrO involved in electron transfer or repaired by H2ur − cannot be determined by the techniques used in this study. It is possible that more than one Trp–Tyr pair is involved in the electron transfer and more than one radical reacts with H2ur − with similar rates. However, we confirmed that the initial radical site on the protein can rapidly relocate to another site through intra- or intermolecular reactions and showed that, depending on the protein and its concentration, H2ur − can inhibit this process. H2ur − and Hasc −, both significant endogenous antioxidants, are thus both able to repair protein radicals. H2ur − would be expected to repair protein radicals faster than Hasc − (a) in tissues or compartments that contain H2ur − at higher concentrations than Hasc −, such as in plasma, heart, and saliva [11,13–17,56], and (b) under conditions where Hasc − is unstable, e.g., at the acidic pH of the stomach [57]. We also show here that these processes depend on the protein. The radical product of Reaction (18), Hur •−, is not expected to oxidize proteins, because at pH 7 the E°′(Hur •−, H +/H2ur −) [28] is lower than those of the amino acids [58] and it does not react with O2 (k b 10 − 2 M − 1 s − 1 [28]). It reacts very fast with O2•− (k = 8.8 × 10 8 M − 1 s − 1 [30]), which leads to H2ur − repair or to products; it can further decay to oxidation products, e.g., by Reaction (5), or it is repaired by several exogenous antioxidants, such as flavonoids [30,38,59], or by Hasc − (k19 = 10 6 M − 1 s − 1 at pH 7 [28]). The repair by Hasc − is in agreement with the standard electrode potentials at pH 7 of the Hur •−/H2ur − and ascorbyl radical (asc •−)/Hasc − couples (+0.59 [28] and +0.28 V [60], respectively): Hur

•−

−

−

•−

þ Hasc →H2 ur þ asc

ð19Þ

The significance of Reaction (19) is clear: in the case in which H2ur − repairs oxidants more rapidly than Hasc −, H2ur − will be repaired, and thus conserved, whereas Hasc − will be consumed. Although we measured only the rate constants of the reactions of H2ur − with Pr(Trp •) and Pr(TyrO •), these should be rather representative for reactions of H2ur − with other amino acid radicals in proteins, so that a general reaction can be written: •

−

Pr þ H2 ur →PrH þ Hur

•−

ð20Þ

In the presence of O2, the repair process of protein radicals by H2ur − and Hasc − would compete with peroxyl radical formation: •

•

Pr þ O2 →PrOO

ð21Þ

Overall, oxidation of proteins would thus be―at least partially― prevented. H2ur − is likely to interfere with the oxidation of proteins also at the next stage of the process, namely by reaction with protein peroxyl radicals: •

−

PrOO þ H2 ur →PrOOH þ Hur

•−

ð22Þ

Indeed, H2ur − is known to react with peroxyl radicals with rate constants on the order of 10 6–10 8 M − 1 s − 1 [61–64]. However, this Reaction (22) does not constitute repair, because the product, a hydroperoxide, is unstable and is likely to propagate damage [65]. In view of the high concentration of H2ur − in living organisms and reactivity toward protein radicals, H2ur − may contribute significantly to the antioxidant defenses of the organism. Acknowledgments This work was supported by the Eidgenossische Technische Hochschule, Zürich, Switzerland, and the Swiss National Foundation.

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