Photochemistry and Photobiology, 1975, Vol. 21, pp. 165-171.
Pergamon Press. Printed in Great Britain
THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS I. B. C. MATHESON,R. D. ETHERIDGE,NANCYR. KRATOWCH and JOHN LEE Department of Biochemistry, University of Georgia, Athens, Georgia 30602, U.S.A.
(Received 23 August 1974; accepted 8 October 1974) Abstract-The physical quenching of singlet molecular oxygen ('Ag) by amino acids and proteins in D20solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1.06 urn outout of a Nd-YAG laser. which absorbs directlv in the electtonic transition 'A, + lu c '2;. The rite of iuenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-bluephotosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in 4 0 , pD 8, are (10' 5? mol-'s-'k alanine 0.2. methionine 3, trvDtouhan 9, histidine 17, carbonic anhydrase 85; lysozyme 150: superoxide dismutase 260, aposuperoxide dismutase 250. INTRODUCTION transition 'A, + 1 v t 'Z[ of the oxygen molecule Dye-sensitized photo-oxidation of the amino acids (Matheson and Lee, 1970). Matheson et al. (1974a) in proteins or the bases in nucleic acids, is believed have recently measured 3 x lo' 2 mol-' s-l for the to be the basis for most of the phenomena classified acceptor rate constant for chemical reaction of the under 'Photodynamic action,' such as the killing of bilirubin anion with singlet oxygen, and this is the cells by illumination in the presence of certain dyes largest acceptor rate constant measured so far for (Blum, 1941; Spikes and Livingston, 1969; Spikes singlet oxygen in solution. This rapid reaction now and MacKnight, 1970). These reactions are usually makes feasible competitive kinetic studies of the carried out in the presence of air, and it is now well interaction of singlet oxygen with other molecules established that the dyes commonly used for the in aqueous solutions. For experimental reasons, the photodynamic processes will also efficiently form solvent must be DO, since H20 has an overtone singlet oxygen ('Ag),by energy-transfer quenching absorption in the region of the laser output, and the of the triplet state of the dye by molecular oxygen lifetime of singlet oxygen is ten times longer in DtO (for review and references, see Kearns, 1971). than in H20(Merkel and Kearns, 1971, 1972). Singlet oxygen is probably an intermediate in some We are able to confirm by direct measurement but not all of these photodynamic reactions (Spikes that, as reported by Nilsson et al. (1973), the amino and MacKnight, 1970), since it reacts rapidly with acids with the highest quenching constants are histidine, tryptophan and methionine (Nilsson et histidine, tryptophan and methionine, but the absolute values for histidine and tryptophan are at., 1973; Nilsson and Kearns, 1973). In a given system the role of singlet oxygen will significantly higher than their estimates. In addition be controlled by competition between its chemical we show that singlet oxygen properties inferred reactivity with an acceptor and the dominant loss from the results of steady-state methylene-blueprocesses. It is the purpose of this work then to photosensitization techniques, may differ, not only report on the rates for physical quenching of singlet quantitatively but also qualitatively, from results oxygen by a number of amino acids, both free in obtained by direct measurement. solution and incorporated within a protein structure. MATERIALS AND METHODS Singlet oxygen can be generated unambiguously The experimental techniques and apparatus for the and at a known rate by absorption of the 1-06 pm singlet oxygen studies have been described previously output of a Nd-YAG laser into the weak electronic (Matheson et al., 1974b). Bovine superoxide dismutase
165
166
1. B. C. MATHESON et a/.
was purified by the method of McCord and Fridovich (1969) and assayed by its ability to inhibit the flavin mononucleotide-ethylenediaminetetra-acetic acid photosensitized reduction of nitro blue tetrazolium (Beauchamp and Fridovitch, 1971). The specific activity of the enzyme was 1.4 x 10' units mg-I, to be compared with 2 x lo' units mg-l published by Beauchamp and Fridovich (1971) using this assay. Copper content was measured as 1.7 mol/mol protein (mol wt 32000). Aposuperoxide dismutase was prepared by dialysis of the native enzyme against three changes of sodium acetate (0.2 M) with EDTA (1 mM, pH 3.8, 24h), followed by water (24h) (McCord and Fridovich, 1969). The lyophilized powder, when redissolved, had a residual superoxide activity of four per cent of the starting material. Carbonic anhydrase was porcine type c and was a gift of R. B. Ashworth. Hen lysozyme and bovine serum albumin (BSA) were from Sigma Chemical Co. (St. Louis, Mo.);other chemicals were of the best commercial grades and were used as supplied. Methylene blue photosensitization was carried out using a Daylight fluorescent lamp after passing through a colored glass filter which removed all light of wavelengths below 600 nm. Absorption measurements were made with a Zeiss PMQII Spectrophotometer. RESULTS
Directly generated singlet oxygen. A kinetic analysis for the generation and decay of singlet oxygen is given in the Appendix; a more detailed presentation is available elsewhere (Matheson et al., 1974b). Singlet oxygen is directly generated by absorption into the 'As+ Iu +'2; electronic transition of molecular oxygen. This is a very weak transition and in order to obtain an observable reaction a high concentration of oxygen and a rapidly reacting acceptor molecule must be used. The high concentration is achieved by applying a high pressure of oxygen, up to 2200 pounds per square inch (psi), over the solution. After vigorous agitation to achieve equilibrium, the oxygen concentrations can be calculated from Henry's Law. The absorption spectrum is too weak to measure directly owing to the low solubility of oxygen in D20, but was assumed to be the same as in Freon (Matheson and Lee, 1971) where the concentration is thirty times higher at this pressure. This assumption is strengthened by the facts that the observed acceptor rate constants for the neutral bilirubin species are the same in Freon and D 2 0 (Matheson et al., 1974a), and that the lifetime for singlet oxygen in D20, derived from measurements of singlet oxygen bilirubin reactions, is in good agreement with that found by Merkel and Kearns (1 972) with their laser-photosensitization technique (Matheson et al., 1974b). The rapidly reacting acceptor molecule used is of course bilirubin, at a pD above 8. Bilirubin may be solubilized in water by adding two equivalents of base per equivalent of bilirubin and diluting into an
appropriate phosphate buffer to achieve a final pD of 8. Care is taken to exclude contamination by H20 at all stages. The use of bilirubin is not straightforward, as it is subject to a number of side reactions, as has been noted (Matheson et a/., 1974a). However with careful control experiments, these difficulties can be overcome. Another problem that arises particularly with the amino acids, is that they are unstable at high oxygen concentrations. For instance, tryptophan at pD 8 and 0.1 M oxygen (1300 psi) in DzO at room temperature, decomposes with a half-life of about 1000 s, as judged by loss of its absorption at 280 nm. This loss i s accompanied by an increase in absorption in the 320 nm region, which suggests that the reaction product is N-formylkynurenine and that this reaction is probably the same free radical autooxidation that has been observed at atmospheric oxygen concentrations (Pailthorpe et a[., 1973; Walrant and Santus, 1974). This reaction with ground-state oxygen makes it impractical to measure the chemical reaction of amino acids with singlet oxygen in our apparatus. The rate of reaction was observed by monitoring the bilirubin absorbance change at 435 nm. The reactions were carried out in D 2 0solution containing EDTA (- 10 mM), equilibrium oxygen pressure 1300 psi, 18 mm path-length cell, 7.0 W C.W. laser irradiation. All reactions were at room temperature (22°C). Steady-state generation of singlet oxygen will result in loss of bilirubin at a pseudo-first-order rate (Appendix). Under the experimental conditions, plots of log OD(435) against time show good linearity, both in the presence and absence of quencher, thus supporting the kinetic assumptions. Suitable corrections are made for loss of bilirubin or quencher by side reactions. The analysfs predicts that the ratio of first-order rate constants in the absence and presence of quencher, k l k ' , should bear a linear relationship to the concentration of added quencher. This is indeed observed, as Fig. 1 shows for methionine. Having
6r
1
2
3
4
5
6
7
8
METHIONINE CONCENTRATION, Q ( p M )
Figure 1. Quenching of singlet oxygen by methionine.
1 67
Singlet oxygen quenchingby amino acids
one of the lower quenching constants, the methionine example should be more subject to the experimental difficulties mentioned. The linearity of the points is seen to be very satisfactory, however. The Appendix also shows that the slope of plots such as that in Fig. 1 is the ratio kQ/k,,from which k ~ the , quenching rate, is calculated by putting k, equal to 5 x lo* s-' (Merkel and Kearns, 1972). Figure 2 shows data for the enzyme bovine
_
I
"/
Table 1. Singlet oxygen physical quenching constants kQ estimated by the direct photophysical method and via methylene blue photosensitization
rc,
(photophys.)
Quencher Histidine Tryptophan Methionine Alanine Superoxide disrnutase Aposuperoxide dismutase Carbonic anhydrase Hen 1ysozyme
kQ (photosens.)
17 9 3 0.2
rc,
(calc.)
5* 4* 5
3* I*
260
82
260
250
110
260
80
65
270
150
-
80
All reactions are in D20,pD 8.1; rate units are 10' 3 mol-' s-'. *From Nilsson et at. (1973) in 1:l water:methanol. 20
SO D
40
60
CONCENTRATION,
80
IW
0 (pM)
Figure 2. Quenching of singlet oxygen by superoxide dismutase. Cross and filled circle correspond to 1 and 7 mM CN-,respectively.
superoxide dismutase as a quencher. Preliminary results of the effect of this enzyme on the oxidation of bilirubin by singlet oxygen have been presented previously (Lee et al., 1973). Superoxide dismutase contains copper at the active site for the dismutase 2 + H202, and this acreaction, 02-+ 02-+ 2H++ 0 tivity is strongly inhibited by cyanide ion, presumably by its strong complexing affinity for the copper. The cross and filled circle in Fig. 2 are for the reaction in the presence of 1 mM and 7 mM cyanide concentration, respectively, and it is seen that there is no significant effect on the quenching. Half quenching of the dismutase reaction is achieved by only 50 p M cyanide. Even when the copper is entirely removed to form the apoenzyme, the quenching rate is unchanged (Table 1). Therefore the copper active site cannot be involved in the strong activity of superoxide dismutase for quenching singlet oxygen. Table 1 shows the collected results for the amino acids and proteins examined, except bovine serum albumin. The kQ estimated both from the photophysical excitation technique with the Nd-YAG Laser and from the methylene blue photosensitized method, either by laser pulse (Nilsson et al., 1973) or steady state, are compared in the first two columns. The kQ (calc.) are estimated by summing the contribution of the histidine, methionine and tryptophan (first column), assuming all other amino acids have no effect, which seems reasonable since their amino group would be tied up in formation of
the peptide bond. This approximation may lead to an underestimate of k0 (calc.) but probably not by more than a factor of two. The results suggest that the histidine, tryptophan and methionine in these proteins must be (within this rough factor of two) as accessible for singlet oxygen as when they are free in solution. When the arginine contribution to lysozyme can be taken into account, it will be expected to enhance the k0 (calc.) significantly. Carbonic anhydrase however appears to be significantly low and the reasons for this are not clear. It has the largest histidine content and the hydrophilic nature of this moiety would be expected to result in their being in contact with solvent. A sample of carbonic anhydrase from the Sigma Chemical Co. gave an even lower result; there may be impurity problems here, and this case needs further study. Figure 3 shows the data for quenching by bovine serum albumin (BSA), and applying the same analysis kQ comes out to be 2.5 x 10'' 2 mol-' s-I, an impossible result since it exceeds the encounter
B SA
CONCENTRATION,
Q (pM)
Figure 3. Apparent quenching of singlet oxygen by bovine serum albumin.
I. B. C. MATHESONet al.
168
limited rate by at least an order of magnitude. This artifact arises from the well known binding of bilirubin to BSA and, if the assumption is made that only free bilirubin (B) reacts with singlet oxygen, and that to a first approximation, albumin-bound bilirubin has the same absorbance as free bilirubin, then the data of Fig. 3 can be analyzed to derive the equilibrium binding constant.
BSA
+ B e BSA - B, K = [BSA - B ] / [ B S A ] [ B ] .
The [ B ]will be obtained from the initial concentration [B10, where [BSA 3 S [B10.
[ B l = [B]o/(l+ K [ B S A I ) . The slope (Fig. 3 ) = K = 5 x lo6 M-',in good agreement with the stronger binding constant (5 x lo6 M-') obtained in equilibrium studies (Beaven et al., 1973). It is important therefore to establish that binding artifacts are not interfering with the k~ measurements made with the other proteins. Binding of the sensitizing dyes to proteins is a well known phenomenon, for instance as has been recently shown for the dye eosin and lysozyme (Kepka and Grossweiner, 1973). Binding of bilirubin to superoxide dismutase was studied both by gel filtration (Sephadex G-25) and equilibrium dialysis. No binding could be detected, which means that there is no binding constant of magnitude greater than lo4 M - ' , and therefore no effect on the estimate of k~ under the conditions of measurement. Methytene blue photosensitized oxidation. The analysis in the Appendix also applies to the methylene blue photosensitized generation of singlet oxygen except that the reaction is complicated by the presence of methylene blue triplets and derived radicals. Nevertheless, the change in bilirubin concentration with time shows good first-order behavior (Fig. 4). These reactions were carried out in a 3 m 2 total volume of D20 solution of final concentrations of potassium phosphate 8 mM, sodium bilirubinate 2.4 p M , methylene blue 0.67 p M and the protein. Stock solutions were made up in H 2 0making dilutions in such a way that the final H20 proportion was less than one per cent. Figure 4 shows that added superoxide dismutase inhibits the bilirubin oxidation. In contrast to the results of the direct experiment (Fig. 2), cyanide ion addition is seen to counter the superoxide dismutase inhibition. Then, if it were not for the direct
"'L 06
200
100
300
400
TIME (5)
Figure 4. Methylene-blue-photosensitized oxidation of bilirubin in D,O, pD 8. First-order decay of bilirubin absorbance, -0-0-0control, 4-04- in presence of superoxide dismutase, -X-X-Xin presence of superoxide dismutase and 170 pMcyanide.
experiments above, one would be led to the false conclusion that the active site copper is involved with the singlet oxygen quenching. The aposuperoxide dismutase shows the same quenching as superoxide dismutase in the methylene blue experiment, reaffirming that this conclusion is incorrect. The origin of the cyanide artifact is not understood: cyanide at these concentrations has no effect on the rate of oxidation of bilirubin in the absence of added quencher. Figure 5 shows the quenching plots for superoxide dismutase both in H 2 0 and D20. The effect of
14
I2
-1
:
1
p
4
2
20
SOD
40
L 60
80
CONCENTRATION, Q (+M)
Figure 5 . Superoxide dismutase quenching of singlet oxygen produced by methylene blue photosensitization. 444- 90,pD 8 ; --cCC DZO, pD 8, SUperoxide dismutase apoenzyme; -X-X-XH,O pH 8.
the longer lifetime of singlet oxygen in DzO is clearly seen by the greater effectiveness of the superoxide dismutase in D20. The ko is estimated from the slope using the k, values from Merkel and Kearns (1972). For H20, k~ = 500 x lo7Y mol-' s", which is six times that for D20(Table 1). The
Singlet oxygen quenching by amino acids lifetime of singlet oxygen in water is therefore shortened to such an extent that other reaction pathways are now dominant, and the superoxide dismutase is effectively intercepting these. Some qualitative statements at least can be made for the methylene blue D20 data, that aposuperoxide dismutase and the holoenzyme have the same quenching ability and that carbonic anhydrase is not significantly lower, as it appears to be for photophysically generated singlet oxygen. The comparison between the directly generated singlet oxygen results and those obtained by photosensitization shows that the latter method is not reliable for quantitative estimates, and even some of the more qualitative conclusions can be open to question.
169
histidine is readily photo-oxidized and the protein loses its ability to reconstitute to the holoenzyme. The holoenzyme is resistant to photo-oxidation however (Forman et al., 1973). Table 1 shows that the quenching ability of proteins is more or less equal to the sum of the constitutive amino acids, histidine, methionine and tryptophan. This means that singlet oxygen can diffuse to all regions of the protein structure with equal facility. Lakowicz and Weber (1973) have come to the same conclusion in regard to ground state .molecular oxygen. They found that every tryptophan molecule in the proteins studied had its fluorescence quenched with equal effectiveness by molecular oxygen. Thus, if the singlet oxygen kQ is a reflection of the chemical acceptor rate, then it DISCUSSION can be concluded that all amino acids in a protein The agreement between the kQ for methionine should be equally accessible to singlet oxygen obtained here and that of Nilsson et al. (1973) reaction. The results of photo-sensitized oxidation would suggest that the three-times-higher result we of proteins clearly show that certain histidines are find for histidine might reflect the nature of the much more reactive than others, suggesting that the quenching species involved. Because of the solubil- photo-oxidizing species must be a large molecule, ity limitations of the acceptor diphenylisobenzofu- such as a sensitizer derived radical, which is ran, Nilsson et al. (1973) carried out their reactions sterically hindered in its approach to the relatively in a solvent composed of 1 : 1, water: methanol. buried groups in the protein. Histidine has a pK of 6.0 (imidazole) and therefore Lakowicz and Weber (1973) prefer to interpret is mostly in the cationic form in this solvent their fluorescence results in terms of rapid fluctuamixture. Its photosensitized oxidation quantum tions of the protein structure, whereby all groups yield in water exhibits a marked decrease below pH are exposed to the solvent some of the time. 6 (Weil, 1965) and this may be a reflection of the However, it would seem that the suggestion of the physical quenching ability. On the other hand, facile diffusion of oxygen would be consistent both tryptophan also exhibits a higher result and a with their results and the present results, as well as different species argument cannot be applied here, accounting for the photo-oxidation inaccessibility since its pK’s are 2.4 and 9.4. Further, if the ko for of some groups. the proteins are calculated by summing the Nilsson The question might be raised as to why bilirubin et a/. (1973) data for the amino acids, the results are becomes ‘inaccessible’ to singlet oxygen oxidation carbonic anhydrase 115 and superoxide dismutase when bound to BSA. It can be suggested that 99, in reasonable agreement with the results from bilirubin is bound in the neutral form, which has an steady state photosensitized oxidation in Table 1. It acceptor rate two hundred times lower than that of is difficult to avoid the conclusion that there is a the anionic species (Matheson et al., 1974a). significant discrepancy between these two sets of Although the quenching of singlet oxygen by data. superoxide dismutase has previously been reported Methylene-blue-photosensitized oxidation of (Lee et al., 1973), two letters have appeared proteins has often been used to show the accessibil- recently claiming superoxide dismutase does not ity of certain of its amino acids to the solvent. For quench singlet oxygen. Bard and Mayeda (1974) instance, two of the histidines in phosphoglucomut- generated singlet oxygen by dismutation of 02ase are fourteen times more susceptible to photo- produced by electrolytic reduction of oxygen, and oxidation than the others (Ray and Koshland, found SOD inhibited the singlet oxygen reaction of 1962). The interpretation was that certain of the 1,3-diphenyIisobenzofuran.They attributed this to amino acids were ‘buried’ within the structure of interference with the dismutation reaction producthe protein and therefore inaccessible to external ing singlet oxygen. Schaap et al. (1974) produced oxidizing agents. A recent study of the methylene singlet oxygen by photosensitization with a dye blue photosensitized oxidation of superoxide dis- attached to a polymer and observed no superoxide rnutase has shown that, with the apoenzyme, dismutase effect on the rate of an acceptor reaction.
170
I. B. C. MATHESONet al.
In this case (in HzO) the superoxide dismutase concentration used (lo-' M) may have been too low for it to compete efficiently with k, for singlet oxygen. There have been a number of reports of the ability to superoxide dismutase to quench low level chemiluminescence from a number of reactions, and this has been interpreted as evidence for a singlet oxygen quenching activity of superoxide dismutase. In these reactions, such as the xanthine-xanthine oxidase oxidation, the chemiluminescence emitting species is not known, although there have been speculations that the emission comes from singlet oxygen (Arneson, 1970). However, the work of Finazzi-Agro et al. (1972), Weser and Paschen (1972) and Paschen and Weser (1973), shows a chemiluminescence quenching at superoxide dismutase concentrations of the order of 10 nM which, if it results from a quenching of singlet oxygen, would have to compete with the solvent loss rate k,. For 50 per cent quenching and in water, k, = 5 x 10' = x s-l, or ko = 5 x 10'' S mol-' s-l, clearly an impossibility, and implying that the species being quenched is much longerlived than singlet oxygen; doubtless it is 02-. That high concentrations of superoxide dismutase can affect singlet oxygen is demonstrated unambiguously by the present observations. Its activity is simply a result of its amino acid content, contributed to mainly by histidine, and is not connected to its catalytic ability for the dismutase reaction, which it catalyzes coincidentally with about the same rate constant, 2 x lo9 S mol-I s-l (Klug et al., 1972; Rotilio et al., 1972). Finally, a quantitative measurement of the singlet oxygen quenching parameters of these amino acids provides a limit to the role singlet oxygen may be suggested to play in living processes. In biological tissue the concentrations of histidine and tryptophan are in the range lo-* M, which will quench singlet oxygen at an overall rate of more than lo6s-' and will be the dominant quenching mode, since the solvent is water with k, = 5 x 10' s-I. For the singlet oxygen to be efficiently intercepted in a process it would need to react with an acceptor present at an effective concentration of at least lo-' M and with a chemical rate constant of the order of lo9 S mol-' s-I. As far as has been measured, bilirubin would be the sole candidate. A protective role against damage by singlet oxygen generated by excited chlorophyll has been suggested as the possible role of p -carotene in photosynthetic organisms (Foote et al., 1970). The k~ for p-carotene in Freon is 3 x lo9 2 mol-' s-' (Matheson et al., 1974b). It is
possible that p-carotene could be present in sufficient concentration in lipid regions to protect against singlet oxygen with an efficiency comparable to that effected by proteins. The same considerations can be applied to experiments which purport to discount the role of singlet oxygen in a biological system. Sternson and Wiley (1972) argued against the participation of singlet oxygen in microsomal oxidations by the failure of added p-carotene to affect the rate of substrate oxidation. Clearly the level of addition of quencher was insufficient to compete with the strong protein effect. A very low level of chemiluminescence from living cells has been observed by a number of workers and attributed to the presence of singlet oxygen. Allen et al. (1972) have reported that this chemiluminescence is enhanced in white blood cells by phagocytosing activity. Considering the very slow rate of the emission from singlet oxygen via the dimol process, 3 x lo-' 9 mol-' s-' (Matheson et al., 1974c) and the lifetime lo-' s calculated above, the organism would need to generate singlet oxygen at an unreasonably high rate to give rise to a detectable emission. Acknowledgement-This work was supported by grants from the National Science Foundation (GP-38218x1) and the National Institutes of Health (HD-07714-01). APPENDIX
Assume singlet oxygen ('Ae) to be formed at a constant rate R , Eq. 1. It will be removed by the physical quenching processes with oxygen, Eq. 2, with the solvent, Eq. 3, and with added quencher Q, Eq. 4. The process is monitored by its chemical reaction with bilirubin anion B which results in absorption change (435 nm).
R
O,+A A +0 2 + 2 0 2 A+Oz A + Q+
0 2
+Q
A+B+B02
(1)
k02
(2)
k,
(3)
kQ
(4)
k,
(5)
The steady-state assumption gives
!!! = 0 = R - A(kozOz+ k, + k,,B + k Q Q ) dt A==R(k, +kQQ)-' since ko, = 2.6 X lo3 2' mol-' s-' (Matheson et a/., 1974b) O2 0.1 M (in D20), k, = 5 x lo' s-', k, = 3 x lo9 3 mol-' s-', B M,kQ lo7 2' mol-' s-', (2 > lO-'M.
-
-
-
Singlet oxygen quenching by amino acids line; if at Q = 0 it has a slope k and k ' for Q = Q, then
or d In B = k.R dt
(k, + k&-'
A plot of log OD(435) with time should be a straight
and kQ can be obtained from this relationship.
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Pergamon Press. Printed in Great Britain
THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS I. B. C. MATHESON,R. D. ETHERIDGE,NANCYR. KRATOWCH and JOHN LEE Department of Biochemistry, University of Georgia, Athens, Georgia 30602, U.S.A.
(Received 23 August 1974; accepted 8 October 1974) Abstract-The physical quenching of singlet molecular oxygen ('Ag) by amino acids and proteins in D20solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1.06 urn outout of a Nd-YAG laser. which absorbs directlv in the electtonic transition 'A, + lu c '2;. The rite of iuenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-bluephotosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in 4 0 , pD 8, are (10' 5? mol-'s-'k alanine 0.2. methionine 3, trvDtouhan 9, histidine 17, carbonic anhydrase 85; lysozyme 150: superoxide dismutase 260, aposuperoxide dismutase 250. INTRODUCTION transition 'A, + 1 v t 'Z[ of the oxygen molecule Dye-sensitized photo-oxidation of the amino acids (Matheson and Lee, 1970). Matheson et al. (1974a) in proteins or the bases in nucleic acids, is believed have recently measured 3 x lo' 2 mol-' s-l for the to be the basis for most of the phenomena classified acceptor rate constant for chemical reaction of the under 'Photodynamic action,' such as the killing of bilirubin anion with singlet oxygen, and this is the cells by illumination in the presence of certain dyes largest acceptor rate constant measured so far for (Blum, 1941; Spikes and Livingston, 1969; Spikes singlet oxygen in solution. This rapid reaction now and MacKnight, 1970). These reactions are usually makes feasible competitive kinetic studies of the carried out in the presence of air, and it is now well interaction of singlet oxygen with other molecules established that the dyes commonly used for the in aqueous solutions. For experimental reasons, the photodynamic processes will also efficiently form solvent must be DO, since H20 has an overtone singlet oxygen ('Ag),by energy-transfer quenching absorption in the region of the laser output, and the of the triplet state of the dye by molecular oxygen lifetime of singlet oxygen is ten times longer in DtO (for review and references, see Kearns, 1971). than in H20(Merkel and Kearns, 1971, 1972). Singlet oxygen is probably an intermediate in some We are able to confirm by direct measurement but not all of these photodynamic reactions (Spikes that, as reported by Nilsson et al. (1973), the amino and MacKnight, 1970), since it reacts rapidly with acids with the highest quenching constants are histidine, tryptophan and methionine (Nilsson et histidine, tryptophan and methionine, but the absolute values for histidine and tryptophan are at., 1973; Nilsson and Kearns, 1973). In a given system the role of singlet oxygen will significantly higher than their estimates. In addition be controlled by competition between its chemical we show that singlet oxygen properties inferred reactivity with an acceptor and the dominant loss from the results of steady-state methylene-blueprocesses. It is the purpose of this work then to photosensitization techniques, may differ, not only report on the rates for physical quenching of singlet quantitatively but also qualitatively, from results oxygen by a number of amino acids, both free in obtained by direct measurement. solution and incorporated within a protein structure. MATERIALS AND METHODS Singlet oxygen can be generated unambiguously The experimental techniques and apparatus for the and at a known rate by absorption of the 1-06 pm singlet oxygen studies have been described previously output of a Nd-YAG laser into the weak electronic (Matheson et al., 1974b). Bovine superoxide dismutase
165
166
1. B. C. MATHESON et a/.
was purified by the method of McCord and Fridovich (1969) and assayed by its ability to inhibit the flavin mononucleotide-ethylenediaminetetra-acetic acid photosensitized reduction of nitro blue tetrazolium (Beauchamp and Fridovitch, 1971). The specific activity of the enzyme was 1.4 x 10' units mg-I, to be compared with 2 x lo' units mg-l published by Beauchamp and Fridovich (1971) using this assay. Copper content was measured as 1.7 mol/mol protein (mol wt 32000). Aposuperoxide dismutase was prepared by dialysis of the native enzyme against three changes of sodium acetate (0.2 M) with EDTA (1 mM, pH 3.8, 24h), followed by water (24h) (McCord and Fridovich, 1969). The lyophilized powder, when redissolved, had a residual superoxide activity of four per cent of the starting material. Carbonic anhydrase was porcine type c and was a gift of R. B. Ashworth. Hen lysozyme and bovine serum albumin (BSA) were from Sigma Chemical Co. (St. Louis, Mo.);other chemicals were of the best commercial grades and were used as supplied. Methylene blue photosensitization was carried out using a Daylight fluorescent lamp after passing through a colored glass filter which removed all light of wavelengths below 600 nm. Absorption measurements were made with a Zeiss PMQII Spectrophotometer. RESULTS
Directly generated singlet oxygen. A kinetic analysis for the generation and decay of singlet oxygen is given in the Appendix; a more detailed presentation is available elsewhere (Matheson et al., 1974b). Singlet oxygen is directly generated by absorption into the 'As+ Iu +'2; electronic transition of molecular oxygen. This is a very weak transition and in order to obtain an observable reaction a high concentration of oxygen and a rapidly reacting acceptor molecule must be used. The high concentration is achieved by applying a high pressure of oxygen, up to 2200 pounds per square inch (psi), over the solution. After vigorous agitation to achieve equilibrium, the oxygen concentrations can be calculated from Henry's Law. The absorption spectrum is too weak to measure directly owing to the low solubility of oxygen in D20, but was assumed to be the same as in Freon (Matheson and Lee, 1971) where the concentration is thirty times higher at this pressure. This assumption is strengthened by the facts that the observed acceptor rate constants for the neutral bilirubin species are the same in Freon and D 2 0 (Matheson et al., 1974a), and that the lifetime for singlet oxygen in D20, derived from measurements of singlet oxygen bilirubin reactions, is in good agreement with that found by Merkel and Kearns (1 972) with their laser-photosensitization technique (Matheson et al., 1974b). The rapidly reacting acceptor molecule used is of course bilirubin, at a pD above 8. Bilirubin may be solubilized in water by adding two equivalents of base per equivalent of bilirubin and diluting into an
appropriate phosphate buffer to achieve a final pD of 8. Care is taken to exclude contamination by H20 at all stages. The use of bilirubin is not straightforward, as it is subject to a number of side reactions, as has been noted (Matheson et a/., 1974a). However with careful control experiments, these difficulties can be overcome. Another problem that arises particularly with the amino acids, is that they are unstable at high oxygen concentrations. For instance, tryptophan at pD 8 and 0.1 M oxygen (1300 psi) in DzO at room temperature, decomposes with a half-life of about 1000 s, as judged by loss of its absorption at 280 nm. This loss i s accompanied by an increase in absorption in the 320 nm region, which suggests that the reaction product is N-formylkynurenine and that this reaction is probably the same free radical autooxidation that has been observed at atmospheric oxygen concentrations (Pailthorpe et a[., 1973; Walrant and Santus, 1974). This reaction with ground-state oxygen makes it impractical to measure the chemical reaction of amino acids with singlet oxygen in our apparatus. The rate of reaction was observed by monitoring the bilirubin absorbance change at 435 nm. The reactions were carried out in D 2 0solution containing EDTA (- 10 mM), equilibrium oxygen pressure 1300 psi, 18 mm path-length cell, 7.0 W C.W. laser irradiation. All reactions were at room temperature (22°C). Steady-state generation of singlet oxygen will result in loss of bilirubin at a pseudo-first-order rate (Appendix). Under the experimental conditions, plots of log OD(435) against time show good linearity, both in the presence and absence of quencher, thus supporting the kinetic assumptions. Suitable corrections are made for loss of bilirubin or quencher by side reactions. The analysfs predicts that the ratio of first-order rate constants in the absence and presence of quencher, k l k ' , should bear a linear relationship to the concentration of added quencher. This is indeed observed, as Fig. 1 shows for methionine. Having
6r
1
2
3
4
5
6
7
8
METHIONINE CONCENTRATION, Q ( p M )
Figure 1. Quenching of singlet oxygen by methionine.
1 67
Singlet oxygen quenchingby amino acids
one of the lower quenching constants, the methionine example should be more subject to the experimental difficulties mentioned. The linearity of the points is seen to be very satisfactory, however. The Appendix also shows that the slope of plots such as that in Fig. 1 is the ratio kQ/k,,from which k ~ the , quenching rate, is calculated by putting k, equal to 5 x lo* s-' (Merkel and Kearns, 1972). Figure 2 shows data for the enzyme bovine
_
I
"/
Table 1. Singlet oxygen physical quenching constants kQ estimated by the direct photophysical method and via methylene blue photosensitization
rc,
(photophys.)
Quencher Histidine Tryptophan Methionine Alanine Superoxide disrnutase Aposuperoxide dismutase Carbonic anhydrase Hen 1ysozyme
kQ (photosens.)
17 9 3 0.2
rc,
(calc.)
5* 4* 5
3* I*
260
82
260
250
110
260
80
65
270
150
-
80
All reactions are in D20,pD 8.1; rate units are 10' 3 mol-' s-'. *From Nilsson et at. (1973) in 1:l water:methanol. 20
SO D
40
60
CONCENTRATION,
80
IW
0 (pM)
Figure 2. Quenching of singlet oxygen by superoxide dismutase. Cross and filled circle correspond to 1 and 7 mM CN-,respectively.
superoxide dismutase as a quencher. Preliminary results of the effect of this enzyme on the oxidation of bilirubin by singlet oxygen have been presented previously (Lee et al., 1973). Superoxide dismutase contains copper at the active site for the dismutase 2 + H202, and this acreaction, 02-+ 02-+ 2H++ 0 tivity is strongly inhibited by cyanide ion, presumably by its strong complexing affinity for the copper. The cross and filled circle in Fig. 2 are for the reaction in the presence of 1 mM and 7 mM cyanide concentration, respectively, and it is seen that there is no significant effect on the quenching. Half quenching of the dismutase reaction is achieved by only 50 p M cyanide. Even when the copper is entirely removed to form the apoenzyme, the quenching rate is unchanged (Table 1). Therefore the copper active site cannot be involved in the strong activity of superoxide dismutase for quenching singlet oxygen. Table 1 shows the collected results for the amino acids and proteins examined, except bovine serum albumin. The kQ estimated both from the photophysical excitation technique with the Nd-YAG Laser and from the methylene blue photosensitized method, either by laser pulse (Nilsson et al., 1973) or steady state, are compared in the first two columns. The kQ (calc.) are estimated by summing the contribution of the histidine, methionine and tryptophan (first column), assuming all other amino acids have no effect, which seems reasonable since their amino group would be tied up in formation of
the peptide bond. This approximation may lead to an underestimate of k0 (calc.) but probably not by more than a factor of two. The results suggest that the histidine, tryptophan and methionine in these proteins must be (within this rough factor of two) as accessible for singlet oxygen as when they are free in solution. When the arginine contribution to lysozyme can be taken into account, it will be expected to enhance the k0 (calc.) significantly. Carbonic anhydrase however appears to be significantly low and the reasons for this are not clear. It has the largest histidine content and the hydrophilic nature of this moiety would be expected to result in their being in contact with solvent. A sample of carbonic anhydrase from the Sigma Chemical Co. gave an even lower result; there may be impurity problems here, and this case needs further study. Figure 3 shows the data for quenching by bovine serum albumin (BSA), and applying the same analysis kQ comes out to be 2.5 x 10'' 2 mol-' s-I, an impossible result since it exceeds the encounter
B SA
CONCENTRATION,
Q (pM)
Figure 3. Apparent quenching of singlet oxygen by bovine serum albumin.
I. B. C. MATHESONet al.
168
limited rate by at least an order of magnitude. This artifact arises from the well known binding of bilirubin to BSA and, if the assumption is made that only free bilirubin (B) reacts with singlet oxygen, and that to a first approximation, albumin-bound bilirubin has the same absorbance as free bilirubin, then the data of Fig. 3 can be analyzed to derive the equilibrium binding constant.
BSA
+ B e BSA - B, K = [BSA - B ] / [ B S A ] [ B ] .
The [ B ]will be obtained from the initial concentration [B10, where [BSA 3 S [B10.
[ B l = [B]o/(l+ K [ B S A I ) . The slope (Fig. 3 ) = K = 5 x lo6 M-',in good agreement with the stronger binding constant (5 x lo6 M-') obtained in equilibrium studies (Beaven et al., 1973). It is important therefore to establish that binding artifacts are not interfering with the k~ measurements made with the other proteins. Binding of the sensitizing dyes to proteins is a well known phenomenon, for instance as has been recently shown for the dye eosin and lysozyme (Kepka and Grossweiner, 1973). Binding of bilirubin to superoxide dismutase was studied both by gel filtration (Sephadex G-25) and equilibrium dialysis. No binding could be detected, which means that there is no binding constant of magnitude greater than lo4 M - ' , and therefore no effect on the estimate of k~ under the conditions of measurement. Methytene blue photosensitized oxidation. The analysis in the Appendix also applies to the methylene blue photosensitized generation of singlet oxygen except that the reaction is complicated by the presence of methylene blue triplets and derived radicals. Nevertheless, the change in bilirubin concentration with time shows good first-order behavior (Fig. 4). These reactions were carried out in a 3 m 2 total volume of D20 solution of final concentrations of potassium phosphate 8 mM, sodium bilirubinate 2.4 p M , methylene blue 0.67 p M and the protein. Stock solutions were made up in H 2 0making dilutions in such a way that the final H20 proportion was less than one per cent. Figure 4 shows that added superoxide dismutase inhibits the bilirubin oxidation. In contrast to the results of the direct experiment (Fig. 2), cyanide ion addition is seen to counter the superoxide dismutase inhibition. Then, if it were not for the direct
"'L 06
200
100
300
400
TIME (5)
Figure 4. Methylene-blue-photosensitized oxidation of bilirubin in D,O, pD 8. First-order decay of bilirubin absorbance, -0-0-0control, 4-04- in presence of superoxide dismutase, -X-X-Xin presence of superoxide dismutase and 170 pMcyanide.
experiments above, one would be led to the false conclusion that the active site copper is involved with the singlet oxygen quenching. The aposuperoxide dismutase shows the same quenching as superoxide dismutase in the methylene blue experiment, reaffirming that this conclusion is incorrect. The origin of the cyanide artifact is not understood: cyanide at these concentrations has no effect on the rate of oxidation of bilirubin in the absence of added quencher. Figure 5 shows the quenching plots for superoxide dismutase both in H 2 0 and D20. The effect of
14
I2
-1
:
1
p
4
2
20
SOD
40
L 60
80
CONCENTRATION, Q (+M)
Figure 5 . Superoxide dismutase quenching of singlet oxygen produced by methylene blue photosensitization. 444- 90,pD 8 ; --cCC DZO, pD 8, SUperoxide dismutase apoenzyme; -X-X-XH,O pH 8.
the longer lifetime of singlet oxygen in DzO is clearly seen by the greater effectiveness of the superoxide dismutase in D20. The ko is estimated from the slope using the k, values from Merkel and Kearns (1972). For H20, k~ = 500 x lo7Y mol-' s", which is six times that for D20(Table 1). The
Singlet oxygen quenching by amino acids lifetime of singlet oxygen in water is therefore shortened to such an extent that other reaction pathways are now dominant, and the superoxide dismutase is effectively intercepting these. Some qualitative statements at least can be made for the methylene blue D20 data, that aposuperoxide dismutase and the holoenzyme have the same quenching ability and that carbonic anhydrase is not significantly lower, as it appears to be for photophysically generated singlet oxygen. The comparison between the directly generated singlet oxygen results and those obtained by photosensitization shows that the latter method is not reliable for quantitative estimates, and even some of the more qualitative conclusions can be open to question.
169
histidine is readily photo-oxidized and the protein loses its ability to reconstitute to the holoenzyme. The holoenzyme is resistant to photo-oxidation however (Forman et al., 1973). Table 1 shows that the quenching ability of proteins is more or less equal to the sum of the constitutive amino acids, histidine, methionine and tryptophan. This means that singlet oxygen can diffuse to all regions of the protein structure with equal facility. Lakowicz and Weber (1973) have come to the same conclusion in regard to ground state .molecular oxygen. They found that every tryptophan molecule in the proteins studied had its fluorescence quenched with equal effectiveness by molecular oxygen. Thus, if the singlet oxygen kQ is a reflection of the chemical acceptor rate, then it DISCUSSION can be concluded that all amino acids in a protein The agreement between the kQ for methionine should be equally accessible to singlet oxygen obtained here and that of Nilsson et al. (1973) reaction. The results of photo-sensitized oxidation would suggest that the three-times-higher result we of proteins clearly show that certain histidines are find for histidine might reflect the nature of the much more reactive than others, suggesting that the quenching species involved. Because of the solubil- photo-oxidizing species must be a large molecule, ity limitations of the acceptor diphenylisobenzofu- such as a sensitizer derived radical, which is ran, Nilsson et al. (1973) carried out their reactions sterically hindered in its approach to the relatively in a solvent composed of 1 : 1, water: methanol. buried groups in the protein. Histidine has a pK of 6.0 (imidazole) and therefore Lakowicz and Weber (1973) prefer to interpret is mostly in the cationic form in this solvent their fluorescence results in terms of rapid fluctuamixture. Its photosensitized oxidation quantum tions of the protein structure, whereby all groups yield in water exhibits a marked decrease below pH are exposed to the solvent some of the time. 6 (Weil, 1965) and this may be a reflection of the However, it would seem that the suggestion of the physical quenching ability. On the other hand, facile diffusion of oxygen would be consistent both tryptophan also exhibits a higher result and a with their results and the present results, as well as different species argument cannot be applied here, accounting for the photo-oxidation inaccessibility since its pK’s are 2.4 and 9.4. Further, if the ko for of some groups. the proteins are calculated by summing the Nilsson The question might be raised as to why bilirubin et a/. (1973) data for the amino acids, the results are becomes ‘inaccessible’ to singlet oxygen oxidation carbonic anhydrase 115 and superoxide dismutase when bound to BSA. It can be suggested that 99, in reasonable agreement with the results from bilirubin is bound in the neutral form, which has an steady state photosensitized oxidation in Table 1. It acceptor rate two hundred times lower than that of is difficult to avoid the conclusion that there is a the anionic species (Matheson et al., 1974a). significant discrepancy between these two sets of Although the quenching of singlet oxygen by data. superoxide dismutase has previously been reported Methylene-blue-photosensitized oxidation of (Lee et al., 1973), two letters have appeared proteins has often been used to show the accessibil- recently claiming superoxide dismutase does not ity of certain of its amino acids to the solvent. For quench singlet oxygen. Bard and Mayeda (1974) instance, two of the histidines in phosphoglucomut- generated singlet oxygen by dismutation of 02ase are fourteen times more susceptible to photo- produced by electrolytic reduction of oxygen, and oxidation than the others (Ray and Koshland, found SOD inhibited the singlet oxygen reaction of 1962). The interpretation was that certain of the 1,3-diphenyIisobenzofuran.They attributed this to amino acids were ‘buried’ within the structure of interference with the dismutation reaction producthe protein and therefore inaccessible to external ing singlet oxygen. Schaap et al. (1974) produced oxidizing agents. A recent study of the methylene singlet oxygen by photosensitization with a dye blue photosensitized oxidation of superoxide dis- attached to a polymer and observed no superoxide rnutase has shown that, with the apoenzyme, dismutase effect on the rate of an acceptor reaction.
170
I. B. C. MATHESONet al.
In this case (in HzO) the superoxide dismutase concentration used (lo-' M) may have been too low for it to compete efficiently with k, for singlet oxygen. There have been a number of reports of the ability to superoxide dismutase to quench low level chemiluminescence from a number of reactions, and this has been interpreted as evidence for a singlet oxygen quenching activity of superoxide dismutase. In these reactions, such as the xanthine-xanthine oxidase oxidation, the chemiluminescence emitting species is not known, although there have been speculations that the emission comes from singlet oxygen (Arneson, 1970). However, the work of Finazzi-Agro et al. (1972), Weser and Paschen (1972) and Paschen and Weser (1973), shows a chemiluminescence quenching at superoxide dismutase concentrations of the order of 10 nM which, if it results from a quenching of singlet oxygen, would have to compete with the solvent loss rate k,. For 50 per cent quenching and in water, k, = 5 x 10' = x s-l, or ko = 5 x 10'' S mol-' s-l, clearly an impossibility, and implying that the species being quenched is much longerlived than singlet oxygen; doubtless it is 02-. That high concentrations of superoxide dismutase can affect singlet oxygen is demonstrated unambiguously by the present observations. Its activity is simply a result of its amino acid content, contributed to mainly by histidine, and is not connected to its catalytic ability for the dismutase reaction, which it catalyzes coincidentally with about the same rate constant, 2 x lo9 S mol-I s-l (Klug et al., 1972; Rotilio et al., 1972). Finally, a quantitative measurement of the singlet oxygen quenching parameters of these amino acids provides a limit to the role singlet oxygen may be suggested to play in living processes. In biological tissue the concentrations of histidine and tryptophan are in the range lo-* M, which will quench singlet oxygen at an overall rate of more than lo6s-' and will be the dominant quenching mode, since the solvent is water with k, = 5 x 10' s-I. For the singlet oxygen to be efficiently intercepted in a process it would need to react with an acceptor present at an effective concentration of at least lo-' M and with a chemical rate constant of the order of lo9 S mol-' s-I. As far as has been measured, bilirubin would be the sole candidate. A protective role against damage by singlet oxygen generated by excited chlorophyll has been suggested as the possible role of p -carotene in photosynthetic organisms (Foote et al., 1970). The k~ for p-carotene in Freon is 3 x lo9 2 mol-' s-' (Matheson et al., 1974b). It is
possible that p-carotene could be present in sufficient concentration in lipid regions to protect against singlet oxygen with an efficiency comparable to that effected by proteins. The same considerations can be applied to experiments which purport to discount the role of singlet oxygen in a biological system. Sternson and Wiley (1972) argued against the participation of singlet oxygen in microsomal oxidations by the failure of added p-carotene to affect the rate of substrate oxidation. Clearly the level of addition of quencher was insufficient to compete with the strong protein effect. A very low level of chemiluminescence from living cells has been observed by a number of workers and attributed to the presence of singlet oxygen. Allen et al. (1972) have reported that this chemiluminescence is enhanced in white blood cells by phagocytosing activity. Considering the very slow rate of the emission from singlet oxygen via the dimol process, 3 x lo-' 9 mol-' s-' (Matheson et al., 1974c) and the lifetime lo-' s calculated above, the organism would need to generate singlet oxygen at an unreasonably high rate to give rise to a detectable emission. Acknowledgement-This work was supported by grants from the National Science Foundation (GP-38218x1) and the National Institutes of Health (HD-07714-01). APPENDIX
Assume singlet oxygen ('Ae) to be formed at a constant rate R , Eq. 1. It will be removed by the physical quenching processes with oxygen, Eq. 2, with the solvent, Eq. 3, and with added quencher Q, Eq. 4. The process is monitored by its chemical reaction with bilirubin anion B which results in absorption change (435 nm).
R
O,+A A +0 2 + 2 0 2 A+Oz A + Q+
0 2
+Q
A+B+B02
(1)
k02
(2)
k,
(3)
kQ
(4)
k,
(5)
The steady-state assumption gives
!!! = 0 = R - A(kozOz+ k, + k,,B + k Q Q ) dt A==R(k, +kQQ)-' since ko, = 2.6 X lo3 2' mol-' s-' (Matheson et a/., 1974b) O2 0.1 M (in D20), k, = 5 x lo' s-', k, = 3 x lo9 3 mol-' s-', B M,kQ lo7 2' mol-' s-', (2 > lO-'M.
-
-
-
Singlet oxygen quenching by amino acids line; if at Q = 0 it has a slope k and k ' for Q = Q, then
or d In B = k.R dt
(k, + k&-'
A plot of log OD(435) with time should be a straight
and kQ can be obtained from this relationship.
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