Physiological Concentrations of Ascorbate Cannot Prevent the

Article pubs.acs.org/crt

Physiological Concentrations of Ascorbate Cannot Prevent the Potentially Damaging Reactions of Protein Radicals in Humans Thomas Nauser† and Janusz M. Gebicki*,‡ †

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zurich CH8093, Switzerland Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia

‡

ABSTRACT: The principal initial biological targets of free radicals formed under conditions of oxidative stress are the proteins. The most common products of the interaction are carbon-centered alkyl radicals which react rapidly with oxygen to form peroxyl radicals and hydroperoxides. All these species are reactive, capable of propagating the free radical damage to enzymes, nucleic acids, lipids, and endogenous antioxidants, leading finally to the pathologies associated with oxidative stress. The best chance of preventing this chain of damage is in early repair of the protein radicals by antioxidants. Estimate of the effectiveness of the physiologically significant antioxidants requires knowledge of the antioxidant tissue concentrations and rate constants of their reaction with protein radicals. Previous studies by pulse radiolysis have shown that only ascorbate can repair the Trp and Tyr protein radicals before they form peroxyl radicals under physiological concentrations of oxygen. We have now extended this work to other protein C-centered radicals generated by hydroxyl radicals because these and many other free radicals formed under oxidative stress can produce secondary radicals on virtually any amino acid residue. Pulse radiolysis identified two classes of rate constants for reactions of protein radicals with ascorbate, a faster one in the range (9−60) × 107 M−1 s−1 and a slow one with a range of (0.5−2) × 107 M−1 s−1. These results show that ascorbate can prevent further reactions of protein radicals only in the few human tissues where its concentration exceeds about 2.5 mM.

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rate forming peroxyl radicals (PrOO•).7 The PrOO• are potent oxidants with reduction potentials in the range of 0.77−1.44 V, capable of propagating oxidative damage to cell and tissue constituents.7−10 They are readily reduced to hydroperoxides (PrOOH) which can undergo one- and two-electron reactions generating a range of new potentially damaging species.11 These events constitute a chain of deleterious processes whose interruption in vivo is likely to be most effective when applied before the damage becomes widespread, when every new reaction may require separate treatment. This can be accomplished by repair of the Pr• by an agent (AH) able to prevail against O2 in the competition for Pr•:

INTRODUCTION The development of oxidative damage by free radicals and other partially reduced oxygen species (PROS)1 in living organisms occurs in a series of stages initiated by the formation of the reactive agent(s).2,3 In the next stage, the PROS oxidize target molecules in reactions determined by the nature of the oxidant and target and the probability of their reaction. If the initial target is vital for normal functioning of the organism, damage will follow. However, it is more likely that the first target will simply transfer the radical to other cell and tissue components, until the chain reaches a vital target. Damage to this target can then initiate the development of some of the numerous pathologies associated with oxidative stress.4 Informed prevention of these events by some form of antioxidant intervention requires identification of the PROS and their initial targets. Many of the biologically significant free radicals and other oxidants have been identified,5 and there is increasing evidence that the oxidation of proteins, rather than lipids or DNA, is the first step in the chain of damage in membranes, in chromatin and in cells.6 A common consequence of the reaction of proteins with a wide range of biologically relevant single-electron oxidants is the formation of carbon-centered radicals (Pr•).3,4,7,8 The Pr• are a biological hazard because they can give rise to a range of reactive derivatives; under physiological conditions, a high proportion combines with O2 at close to a diffusion-controlled © 2017 American Chemical Society

Pr • + O2 → PrOO•

(1)

Pr • + AH → PrH + A•

(2) •

Under physiological conditions, the fate of the Pr is determined by several factors of which the concentrations of AH and O2 and the rate constants (k) of reactions 1 and 2 are particularly important.10 Reaction 1 is fast, with the k1 value close to 2 × 109 M−1 s−1.7 Successful competition by the AH requires the rate constant k2 to be of similar magnitude, unless its concentration can be made much higher than that of O2. The concentrations of O2 and the main endogenous candidates Received: June 7, 2017 Published: July 26, 2017 1702

DOI: 10.1021/acs.chemrestox.7b00160 Chem. Res. Toxicol. 2017, 30, 1702−1710

Article

Chemical Research in Toxicology for the role of AH, glutathione (GSH), ascorbate (HAsc−), and urate (H2ur−) in tissues are often known.4,12,13 However, as pointed out by Hawkins and Davies, in 2001 there was little mechanistic or kinetic data available on the reaction of proteinderived radicals with antioxidants.3 Since then, some rate constants have been determined for a restricted range of protein radicals by the application of the fast kinetic methods of pulse radiolysis and flash photolysis, with optical detection of the reactive intermediates generated. However, this restricted direct observation of Pr• to the formation of radicals in Trp and Tyr residues and measurements of the acceleration of their decay by different antioxidants. The results provided the values of k2 for reactions of free and protein-bound Trp and Tyr radicals with the antioxidants ascorbate, trolox, urate, GSH, and some flavonoids.2,14−18 Of these, only physiological concentrations of ascorbate and urate were able to give protection from the formation of peroxyl radicals in protein-bound Trp• and TyrO•. However, since Trp and Tyr account for only 1.1 and 2.3% of amino acids in typical proteins19 and have a relatively low probability of location in the protein surface, they are not likely to be the principal initial targets of the most potent physiologically relevant radicals. Our study was based on the premise that it was important to determine if the work on reactions of the protein Trp and Tyr radicals with antioxidants could be extended to other amino acid residues, more likely to be the targets of radicals. We evaluated the ability of physiological concentrations of ascorbate to repair the Pr• generated by the hydroxyl radicals (HO•) by using pulse radiolysis to determine the rate constants of reaction 2 for several proteins. This has not been attempted so far, probably because the derivation of the rate constants of reaction 3 is only possible if the product Pr• is a single kind of protein radical or different radicals reacting at similar rates with the ascorbate. PrH + HO• → Pr • + H 2O

with solutions of proteins because of foaming. The N2O-saturated solutions were then transferred in a gas tight syringe into the irradiation cell through PEEK tubing. Normally, fresh solutions were used for each pulse, and KSCN solutions were used to determine the radiation doses.21 Kinetic analysis of the time-dependent absorbance changes was performed with Excel (Microsoft, USA). Protein Radicals. Under the conditions used, pulse radiolysis generated eaq− and HO• and H• radicals in yields of 2.8, 2.8, and 0.6 mol−7 J −1, respecively.7 In the presence of N2O, the electrons were converted to HO•, doubling its yield.7 Interaction of the HO• with proteins was by abstraction of H from the solvent-accessible surface residues or addition to aromatic residues.10 The rates of many such reactions are some of the fastest measured for the HO•, typically diffusion-controlled, with rate constants close to 1010 M−1 s−1.22

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RESULTS Pulse radiolysis of 100 μM solution of lysozyme containing 10 mM phosphate at pH 7 and saturated with N2O produced transient absorbance at 360 nm (Figure 1). This wavelength

Figure 1. Formation of ascorbyl radicals by lysozyme radicals (Pr•). A solution of 100 μM lysozyme saturated with N2O containing 10 mM phosphate at a final pH of 7.1 was pulse-irradiated in the absence (lower curve) and presence (upper curve) of 50 μM ascorbate. Absorbance was measured at 360 nm.

(3)

Our results demonstrate that the rate constants of reaction 2 could be determined for ascorbate and radicals generated in 5 proteins. They also show that physiological concentrations of ascorbate cannot prevent the formation of the reactive protein peroxyl radicals and their derivatives in most human tissues.

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was used in most experiments because it corresponds to the maximum absorbance of the ascorbyl radical (Asc•−).23 The low energy dose of 3.5 Gy minimized radical−radical recombination. In the absence of ascorbate, the Pr• decay kinetics at 360 nm were not simple, neither pure first nor second order. When the protein solution contained 50 μM ascorbate, the 360 nm absorbance developed immediately after the pulse was much higher and increased over ∼100 μs, with subsequent slow decay (Figure 1, upper curve). This was consistent with the formation of Asc•− in the reaction sequence

MATERIALS AND METHODS

All reagents were of analytical grade obtained from Fluka (Buchs, Switzerland) and from Sigma-Aldrich (Sydney, Australia). The proteins were bovine pancreas insulin, chicken egg white lysozyme, bovine pancreas chymotrypsin type II, chicken egg ovalbumin, and fatty acid free human serum albumin. Solutions were freshly made up in Milli-Q water. Neutral aqueous solutions of ascorbate prepared at 0°, with the concentration monitored at 265 nm, were stable for several hours. While most solutions were buffered with 10 mM phosphate, lysozyme was initially dissolved at pH up to 9 and backtitrated to the final pH of 7.1. Optically clear solutions of insulin required a pH of 9.9. Pulse Radiolysis. Details of the pulse apparatus have been described20 except that the optical path length was increased to 6 cm. In most experiments, the energy doses were kept below 5 Gy (1 Gy = 1 J kg−1) to minimize the possibility of radical−radical reactions. The irradiation cell had a volume of 300 μL. Before irradiation, the solutions were degassed in Schlenk tubes by repeated evacuation and saturation with N2O or by equilibration for several hours in a glovebox under an atmosphere containing less than 4 ppm of dioxygen. These procedures did not result in complete removal of dioxygen, but the residual [O2] was less than 10 μM. Complete removal of the O2 could be achieved by bubbling with an inert gas, but this was not possible

HO• + PrH → H 2O + Pr •

(4)

Pr • + HAsc− → PrH + Asc•−

(5) •

Using published rate constants for the reactions of HO with lysozyme and ascorbate,22 it can be estimated that over 90% of the radicals generated in the presence of 50 μM ascorbate reacted with the protein. Additional details of the formation and decay of the lysozyme and ascorbyl radicals are shown in Figure 2. In the absence of ascorbate (left panel), pulse radiolysis produced transient absorbance of decreasing intensity from 300 to 450 nm which decayed over 200 μs. The absorbance showed no features 1703


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Chemical Research in Toxicology

Figure 2. Time course of the changes in absorbance spectrum of a pulse-irradiated solution of 100 μM lysozyme. Left panel: results in the absence of ascorbate. Middle panel: results in the presence of 50 μM ascorbate. Right panel: net absorbance changes after subtraction of the results shown in the absence from those in the presence of ascorbate. Formation of ascorbyl radicals is shown by absorbance at 360 nm in the middle and right panels, with a tyrosyl radical peak at 410 nm evident in the left and middle panels.

Figure 3. Effect of different concentrations of ascorbate on the kinetics of formation of 360 nm absorbance in pulse irradiated anaerobic solutions of 100 μM solutions of 5 proteins in 10 mM phosphate. Only some of the results are shown for lysozyme, ovalbumin, and human serum albumin, with the additional results included for insulin and chymotrypsin. The final pH of each solution is shown in Table 1.

The results of the extension of these experiments to other proteins and other ascorbate concentrations are illustrated in Figure 3. All solutions contained 100 μM protein, and the results were normalized to a dose of 1 Gy. For lysozyme, ovalbumin, and human serum albumin, the effect of only two ascorbate concentrations are shown, with the full range shown for insulin and chymotrypsin. The general features of the absorbance changes for all the proteins were similar; in the absence of ascorbate, there was only decay, while increasing ascorbate concentrations resulted in increased absorbance and slower decay. The Asc•− peak was reached at ∼100 μM or less after the pulse with all the proteins. Closeness of the maximum absorbance developed in the presence of 100 and 200 μM ascorbate suggested that the relationship between [HAsc−] and

indicative of the presence of identifiable intermediates, except for a prominent peak at 410 nm which is one of two peaks characteristic of the tyrosyl radical (TyrO•).14 The assignment is supported by the observation that formation of the 410 nm peak did not reach a maximum immediately after the pulse but only after about 15 μs, showing that most if not all of the TyrO• was generated by long-range electron transfer from the Trp• formed initially.14 In the presence of 50 μM ascorbate (middle panel), a new absorbance peak formed immediately at 360 nm due to the generation of Asc•−. In the right panel, the absorbance spectrum of the protein alone was subtracted from that recorded in the presence of ascorbate; the graph shows time-dependent formation of Asc•− in agreement with the result shown in Figure 1 and rapid decay of the TyrO• due to its reduction by the ascorbate. 1704


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Figure 4. Analysis of the reaction of insulin radicals in pulse-irradiated solutions containing different concentrations of ascorbate. The solution contained 100 μM insulin and 10 mM phosphate at pH 9.9. Graphs in the left panel show net absorbance after subtraction of the absorbance decay in the absence of ascorbate. In the left panel, the solid lines were generated by a curve-fitting program with one exponential term. The right panel shows the results of the application of a program containing a two-exponential term.

Figure 5. Evidence for 2 simultaneous reactions of insulin radicals with ascorbate. The experimental conditions were as shown in Figure 4. In panel A, the points show the formation of 360 nm absorbance, with the red line indicating the time of the electron pulse. Analysis of the kinetics of formation of the ascorbyl radicals (in B) shows two processes approximately linear with time in the early stages of the reaction. The bottom panels show plots of the pseudo-first-order rate constants of the fast and slow reactions of protein radicals with ascorbate. The experimental point marked × in the slow reaction was omitted from calculation of the bimolecular rate constant k5.

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Chemical Research in Toxicology Table 1. Rate Constants of Reactions of Selected C-Centered Radicals with Ascorbatea radical

Mr (kDa)

pH

k(PrH + HO•) (×1010 M−1 s−1)

k(Pr• + HAsc−) (×107 M−1 s−1)

insulin lysozyme• chymotrypsin• ovalbumin• human serum albumin• N-Ac-Trp•-NH2 lysozyme-Trp• chymotrypsin-Trp• pepsin-Trp• β-lactoglobulin-Trp• insulin-TyrO• lysozyme- TyrO• chymotrypsin- TyrO• pepsin- TyrO• β-lactoglobulin- TyrO•

5.7 14 22.6 45 68

9.9 7.1 6.7 6.2 6.5 7.4 7.0 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4

1.7 4.9 3.7

60, 9 16, (0.5−2) 25, (0.5−2) 11, (0.5−2) 12, (0.5−2) 14 8 16 18 2.2 2.9 1.1 4.0 3.5 0.04

•

7.4 7.4

7.6

ref this this this this this 15 15 15 15 15 15 15 15 15 15

work work work work work

In previous reports, the Trp and Tyr radicals were generated by the azide radical. The (0.5−2) × 107 M−1 s−1 values are estimated for the slow phase of the reaction. a

Figure 6. Kinetics of formation of 360 nm absorbance in pulse-irradiated solutions containing 100 μM chymotrypsin and 10 mM phosphate at a final pH of 6.7 (left panel). Net results are shown after subtraction of the decay of absorbance developed in the absence of ascorbate. The solid lines show computer-generated curves using an equation with a single exponential term. The right panel shows a plot of the values of the pseudo-first order rate constants kobs derived from the initial rates of formation of ascorbyl radicals at different ascorbate concentrations.

at two rates (Figure 5B). When the experiments were repeated with solutions containing different concentrations of HAsc−, the bimolecular rate constants k5 could be derived. The two pseudo-first order reactions with insulin radicals are shown in the plots of kobs in the lower panels. Here kobs = k5 [HAsc−] based on the assumption of an unchanged concentration of the HAsc− following the pulse. The derived bimolecular rate constants are listed in Table 1, where the result marked × (Figure 5 lower right panel) was not used in the calculation. The values of k5 listed for the different proteins have estimated standard deviations of ±15%. Similar analysis was attempted for solutions of 100 μM chymotrypsin (Figure 6). The growth of 360 nm absorbance is shown in the left graph, after subtraction of control decay in the absence of ascorbate. For this protein, the agreement between the observed rates of formation of the Asc•− and the theoretical curve fit using a single exponential term was close for the first 100 μs for 25 and 50 μM HAsc− but deviated at higher concentrations. As in the case of insulin, the deviations indicated the occurrence of at least 2 processes differing in

absorbance was not linear but showed apparent saturation at higher [HAsc−]. Derivation of the rate constants of reaction 5 (k5) for the different protein radicals required computational analysis of the kinetics of formation of the 360 nm absorbance. In the case of insulin, the solutions contained 100 μM insulin and either zero or 50 μM ascorbate, with the final pH of 9.9 ensuring a clear solution. Formation of HAsc− in the pulse-irradiated solutions occurred at a rate which could be analyzed by an equation containing a single exponential factor for only about 70 μs following the pulse (Figure 4, left panel). Deviation of the experimental results from the computed curves was especially large at the higher concentrations of HAsc−. Corrections made by the use of an equation with a double exponential term (Figure 4, right panel) allowed more precise analysis of the reaction kinetics. The red line in Figure 5A indicates the moment of pulse, with subsequent growth of 360 nm absorbance. Analysis of the growth clearly showed three processes: an initial fast “jump” caused by the reaction of the HO• with the insulin, followed by slower buildup of absorbance 1706


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TyrO•; the apparent delay in spontaneous Pr• decay at 340− 370 nm in the presence of HAsc− was due to overlap with absorbance of the Asc•− (Figure 2 middle panel). Derivation of the rate constants of reaction 5 for the different proteins clearly showed the occurrence of at least two processes distinguished by their kinetics, a fast and a slow formation of Asc•− (Figures 4, 5, and 6). This rather unexpected result can be explained by consideration of the mechanism of interaction of HO• with the proteins. The reaction of HO• and many other radicals with globular proteins produces mainly carbon-centered radicals on the amino acid side chains.3 The probability of radical interaction with a particular residue is determined by its frequency of occurrence, side chain surface area, location in solventaccessible site, and the rate constant of reaction 3.3,10,19,22 In our study, one HO• was generated for 50 molecules of each protein, giving the HO• a wide choice of protein sites, producing a range of Pr• likely to react with the HAsc− at different rates. This was not the case for the fast phase of the formation of Asc•−, where we found single values for the rate constants in all the proteins tested (Table 1). At (1.1−6.0) × 108 M−1 s−1, these reactions were unlikely to involve Ccentered radicals whose reduction by HAsc− has normally values lower by 2 orders of magnitude.31,32 The possibility that the fast formation of the Asc•− involved protein peroxyl radicals produced in the presence of up to 10 μM inadvertent O2 can be excluded on kinetic grounds. Generation of Asc•− in reaction 6 with an average of rate constant of 1.5 × 107 M−1 s−1 measured for the reactions of amino acid peroxyl radicals with HAsc− would have a half-life of 200 μs, going to completion in ∼1 ms; this contrasts with results showing consistently that reaction 5 was completed in 100 μs or less for the 5 proteins tested (Figure 3).16

rates. Values of kobs measured for the fast phase gave a linear relationship up to 400 μM [HAsc−], allowing the derivation of the bimolecular rate constant k5 (Figure 6, right panel, and Table 1). However, unlike the results with insulin, it was not possible to obtain a satisfactory fit at longer times after the pulse even by using two exponential terms, allowing only a broad estimate of k5 = (0.5−2) × 107 M−1 s−1 for the slow phase of the reaction of chymotrypsin radical with HAsc− (Table 1). Analysis of the reactions of 200 μM HAsc− with radicals generated in lysozyme, human serum albumin, and ovalbumin showed that the use of a single exponential term corresponded closely to the results only for the first 150 μs, and as in the case of chymotrypsin, attempts to obtain a fit using a double exponential resulted in a large uncertainty for the rate constant of the process recorded between 150 and 450 μs. Moreover, we cannot exclude the possibility that more than two processes were operative. For these proteins, the estimated rate constants for the slow stage were in the same broad range calculated for chymotrypsin (Table 1).

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DISCUSSION Formation and Reactions of Protein Radicals with Ascorbate. The results shown in Figures 1 and 2 demonstrate the feasibility of evaluation of the ability of ascorbate to “repair” the radicals generated in at least one protein. In the case of random attack by the hydroxyl radicals, as applied here, the HO• initially generated primarily carbon-centered radicals.3,7 These have weak absorbance characteristics and decayed quickly in the absence of reactive solutes, mainly in radical− radical reactions (Figures 1 and 2). With some exceptions, the weak absorbance and the general absence of peaks characteristic of identifiable amino acid radicals do not allow measurements of the kinetics of their formation and reactions; exceptions are the Trp and Tyr radicals which absorb at 510 and ∼400 nm.7,24 Additional absorbance at ∼420 nm may be also due to the formation of RSSR•+ radicals generated by HO• in diffusioncontrolled oxidation of solvent-accessible cystine.22,25 In this study, we were particularly interested in the possibility of quantitative kinetic interpretation of the reactions of any protein radicals with ascorbate because it appears to be the only widespread endogenous antioxidant capable of repairing some Pr• under physiological conditions.15 The upper curve in Figure 1 shows that the reaction of HAsc− with such uncharacterized protein radicals can be studied by the kinetics of formation of the Asc•−. More details of the reaction are shown in Figure 2. The presence of transient intermediates was first recorded at 5 μs after the pulse, but the only identifiable radical, TyrO•, appeared after about 20 μs. Clearly, this was not formed directly by the HO•, which disappeared in reaction 4 within 1 μs after the pulse, but by intramolecular long-range electron transfer (LRET) to another amino acid radical attacked by the HO• initially. This slow formation of the TyrO• and its mechanism were reported by Butler et al.,26 and such radical transfer between amino acid residues was confirmed in many studies of peptides and proteins, with first order rate constants of 103 − 104 s−1.24,26−28 A particularly thoroughly studied transfer relevant to this study was between Tyr and Trp• in lysozyme.24,27,29 The general spontaneous decay of absorbance between 300 and 450 nm (Figure 2) was principally due to radical−radical reactions which can result in protein cross-linking and other forms of damage.10,30 The major observable effect in the presence of HAsc− was acceleration of the rate of decay of the

PrOO• + HAsc− → PrOOH + Asc•−

(6)

A mechanism compatible with the faster phase of reaction 5 would involve sulfur and aromatic amino acid radicals, which have sufficiently high rate constants of reaction with HAsc−: 1.2 × 109 M−1 s−1 for Cys•, (0.8−1.6) × 108 M−1 s−1 for proteinTrp•, and (1.7−4.0) × 107 M−1 s−1 for protein-TyrO•.15,32 The validity of this mechanism depends on the product of the surface area and probability of the location of these amino acids in a solvent-accessible part of the protein.33 Analysis of the database of 55 proteins revealed that for Cys, Tyr, and Trp, these products are 1.4%, 3.6%, and 0.8% of the total for the 20 amino acids.19 Since in our study the yield of Asc•− in the fast stage of reaction 5 was 10−20% of the amount of Pr• generated, these values are not sufficient for the expected stoichiometry. Instead, the relatively low amounts of Asc•− formed and the observed rate constants higher than expected for C-centered radicals (Table 1) could be reconciled by transfer of some of the surface radicals reacting slowly with HAsc− to nearby fast reacting Cys, Trp, or Tyr residues by LRET. Such transfer is common in peptides and proteins and involves radical chain reactions, with Tyr, Trp, or Cys as the ultimate sinks for oxidizing equivalents.26,29 Unimolecular radical transfers at 103 s−1 or greater can successfully compete with bimolecular reactions such as 5 with rate constants 106− 107 M−1 s−1 because of the low reactant concentrations. There is also experimental support for the high reactivity of some intramolecular protein Trp and Tyr radicals with HAsc−. A crucial requirement is access by the HAsc−. In our earlier study, the rate constant of 1.4 × 108 M−1 s−1 for the reaction of the N1707


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Chemical Research in Toxicology Ac-Trp• amide free in solution with HAsc− was very close to rate constants of the Trp• located in trypsin, pepsin, and lysozyme, with a lower value for β-lactoglobulin (Table 1). The corresponding values for the TyrO• were lower at (1.1−4.0) × 107 M−1 s−1, again with a lower value for β-lactoglobulin.15 Thus, in many proteins, relocation of a radical from the surface to another residue had little or no effect on the rate of reaction with a relatively large charged molecule. However, as illustrated by β-lactoglobulin, the ease of access is a factor depending on the protein structure. This was also clearly shown in our study of the reaction of insulin-Tyr radicals with HAsc−, which demonstrated that both of the insulin Tyr residues were oxidized by azide radicals to TyrO• but that only one reacted with the HAsc−, presumably because of steric constraints.15 The significance of accessibility is also consistent with quenching of Trp fluorescence by a range of molecules in several proteins.34 The authors found that the ability of small molecules to access Trp residues in nanosecond time scale depended primarily on their location, resulting in classification of the Trp as exposed, moderately accessible, or unexposed. Overall, these observations suggest that the relatively high rate constants of reaction 5 could be explained if they involved cystine, Cys, Tyr, or Trp radicals. Depending on the protein, some of the radicals could be generated in direct very fast reaction with the HO• at the solvent-accessible sites22 and some by radical transfer from any surface amino acid radical to these four amino acids buried and inaccessible to the HO•. The low yield of Asc•− in the fast reactions was probably due to insufficient surface frequency of Cys-Cys, Cys, Tyr, and Trp residues and the inaccessibility of some of their radicals to the HAsc−. This would lead to a loss of Pr• by cross-linking, disproportionation, or decomposition, reducing the amount of Asc•− produced. The very high rate constant of the fast stage of reaction of insulin radical with HAsc− suggests possible involvement of the S-radical generated by the HO• reacting with a solvent-accessible cystine bridge.25 The slow stage of reaction 5 was identified by the inability to model its kinetics using a single exponential term in the analysis. This was especially obvious in the presence of 100 and 200 μM HAsc− after 150 μs following the pulse (Figures 4,5,7). Inclusion of a second exponential allowed the derivation of a rate constant for the slower reaction stage in the case of insulin, but no satisfactory fit could be obtained for the other proteins. On the assumption that the slow reactions were second-order, we estimate their rate constants to be within the rather broad range of (5−20) × 106 M−1 s−1. If the protein radicals involved were C-centered, these values are higher than the k < 106 M−1 s−1 for •CH2OH and k = 1.2 × 106 M−1 s−1 for •C(CH3)2OH reactions with ascorbate.31−33 However, they are reasonably close to the rate constants of reactions of aliphatic amino acid radicals under current study. On the basis of that outlined above, we can assume that the initial protein radicals generated by the HO• will have ∼55% probability of location on the Lys, Ser, Glu, and Asp residues.19 Most of these would be Ccentered, able to react with HAsc− with rate constants of ∼106 M−1 s−1, but the yields of Asc•− are still not sufficient to correspond to the amounts of Pr•. These low yields may be due primarily to LRET from the primary radical sites, with some or most of the final radical sites not accessible to HAsc−. In summary, the kinetic and quantitative aspects of our results allowed the derivation of only approximate values of the reaction rate constants for the slower stage of the reaction of protein radicals with ascorbate. However, these are sufficient to

estimate the limits of the effectiveness of HAsc− in removing potentially damaging Pr• under physiologically relevant conditions, as outlined below (Table 2). These limits depend Table 2. Repair of Protein Radicals in Human Tissues Containing Different Concentrations of Oxygen and Ascorbatea tissue pituitary glands adrenal glands eye lens brain liver spleen pancreas lungs kidney heart muscle testes skeletal muscle thyroid plasma saliva

ascorbate (mM) 4.32− 5.36 3.21− 4.34 2.70− 3.32 1.40− 1.60 1.08− 1.72 1.08− 1.60 1.08− 1.60 0.755 0.53− 1.60 0.53− 1.60 0.32 0.32− 0.43 0.21 0.038− 0.11 0.008− 0.009

oxygen (μM)

[HAsc−] mM for 50% Pr• repairb

Pr• repaired by HAsc−%c

40

1.60

100

40

1.60

100

2.3 ± 0.3

0.09

100

36 ± 2.8

1.44

51−59

44 ± 5.8

1.76

33−100

40

1.60

37−57

40

1.60

36−53

46.2 78 ± 22

1.85 3.12

22 9−27

40

1.60

18−53

40 31.5 ± 1.9

1.60 1.26

11 13−18

40 76

1.60 3.00

7 0.7−2

40

1.60

0.3

a

Concentrations of ascorbate are from ref 12 recalculated using an average value for water content of human tissues of 53%.35 Additional values are found in ref 13. Oxygen concentrations are from ref 36 or assumed to be 40 μM where not reported. Tissue oxygen concentrations were determined at 37°; when a range of values are listed, the average was used. Protein radical repair by ascorbate was calculated with k5 = 5 × 107 M−1 s−1 (Table 1). bConcentrations of HAsc− required for 50% inhibition of reaction Pr• + O2 were estimated by using concentration values making the ratio: rate Pr• + HAsc−/rate Pr• + O2 = 1. cThe % Pr• repaired by HAsc− we estimated with the ratios: rate Pr• + HAsc−/rate Pr• + O2 using concentration values listed in columns 2 and 3.

on the value of the rate constant of reaction 5, which could only be determined within a broad range (Table 1). While the choice of 5 × 107 M−1 s−1 (Table 2) may be considered high, use of any other value within the range measured here would only change the extent of the ability of ascorbate to protect Pr• in human tissues, with lower k5 resulting in lower protection. Biological Significance. Damage to living organisms subjected to oxidative stress has been the subject of intensive studies since the discovery of superoxide dismutase, and the results are documented in thousands of publications (reviewed in ref 4). While much has been discovered, there are some obvious knowledge gaps. This is particularly true at the molecular level, where there is still insufficient quantitative physical−chemical information for distinguishing between likely and unlikely damaging agents, their targets, and the likely and unlikely subsequent reactions in particular situations.36 As a consequence, many of the reactions 1708



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commonly used to account for biological end-points of oxidative stress are not realistic. Much of the relevant information must be derived from thermodynamics, kinetics, and chemical analysis, i.e., from both theory and experiments. Our study was based on evidence demonstrating that proteins are significant targets of free radicals associated with oxidative stress in vivo and that formation of protein radicals can lead to downstream damage.2,3,6,8,30 A positive finding was that a limited range of these radicals may be repaired by physiological concentrations of ascorbate.15 While the HO• is not a primary radical under normal physiological conditions, it can be derived from a wide variety of sources and is widely considered to be the most potent agent responsible for much of the damage associated with oxidative stress.4 It is important to note that the nature of the agent generating the protein radicals is of minor relevance. Studies of the formation of the major derivatives of the Pr•, the hydroperoxides, showed that the precursor C-radicals can be generated by hydroxyl and peroxyl radicals as well as in exposure to the Fenton system (H2O2 + Fe2+), peroxynitrite, xanthine/xanthine oxidase, NADH/NADH oxidase, and activated neutrophils.8 Physiological reality of protein hydroperoxides was confirmed by their formation in cultured cells and in animal and human tissues subjected to oxidative stress.6 Applied to humans, it can be stated with some confidence that protection against damage by the normal physiological concentrations of ascorbate cannot be effective in most tissues (Table 2), supporting the assumption that protein radicals may play an important role in the development of biological damage caused by oxidative stress. More effective protection could in theory be provided by very high tissue ascorbate concentrations achievable by direct infusion, but this involves complex medical input resulting in selective killing of tumor cells, probably by a mechanism involving the generation of H2O2 in the presence of transition metals.13 It seems likely that, particularly under conditions of oxidative stress, antioxidants additional to dietary ascorbate are necessary to enhance the organism’s resistance to damage. The potential effectiveness of other antioxidants in repairing protein radicals is likely to be amenable to testing by the methods used in this study.

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AUTHOR INFORMATION

Corresponding Author

*Department of Biological Sciences, Macquarie University, Balaclava Rd, North Ryde, Sydney, N.S.W. 2109, Australia. Tel: +61 2 9850 8587. E-mail: [email protected]. ORCID

Janusz M. Gebicki: 0000-0002-1619-1716 Funding

This work was partially supported by Macquarie University and the Swiss Federal Institute of Technology. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor D. Günther for making the pulse equipment available for this study.

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ABBREVIATIONS PROS, partially reduced oxygen species; LRET, long range electron transport; AH, antioxidant 1709


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Physiological Concentrations of Ascorbate Cannot Prevent the

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