Jul 6, 1978 - The histidine chemical reaction rate constant shows an increase with pD with a. pK of 6.9. INTRODUCTION. Photosensitized oxidation of amino ...
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Photochemistry ond Pkorobrology. Vol. 29. pp. 879 10 881. 0 Perg.,mun Press Lld. 1979. Printcd in Great Brilmn
CHEMICAL REACTION RATES OF AMINO ACIDS WITH SINGLET OXYGEN I. B. C. MATHESON and JOHNLEE Department of Biochemistry, University of Georgia, Athens, GA 30602, U.S.A. (Received 6 July 1978; accepted 16 October 1978)
Abstract-The chemical reactions of amino acids with singlet oxygen have been measured in D,O solution where the singlet oxygen was generated directly by irradiation of the oxygen 32; + 'Au + l v electronic transaction with the 1.06pm output of an Nd-Yag laser. Chemical reaction was measured as amino acid loss by an amino acid analyzer or by fluorescence in the cases of tryptophan and tyrosine. The chemical rate constants, in units of lo' M - ' s - ' , are histidine 10, tryptophan 3, methionine 1.7. tyrosine 0.8 and alanine 0.2. In the cases of histidine, methionine and alanine the interaction appears to be entirely chemical, i.e. there is no evidence for physical quenching in addition to the chemical reaction. The histidine chemical reaction rate constant shows an increase with pD with a pK of 6.9.
INTRODUCTION
Nilsson et al. (1972) who reported rate constants for Photosensitized oxidation of amino acids and pro- the reaction of amino acids with dye triplet and the teins has been much studied in the past. Early studies chemical reaction and physical quenching rate conWeil et al. (1951) showed that phososensitized oxi- stants for the reaction of amino acids with singlet dation could be used to identify critical amino acid oxygen. residues at the catalytic site in enzymes. The method A method for generating singlet oxygen directly in has been used quite frequently by protein chemists heavy water solution (DzOis used in preference to and has been reviewed most recently by Westhead HzO because of the ten times larger singlet oxygen (1972). An early definitive study on the dye sensitized lifetime, Merkel and Kearns, 1972) by irradiation of photooxidation of the free amino acids was also by the oxygen 3C; -+ 'A + l v electronic transition of Weil (1965) who showed, inter alia, that the rate of oxygen dissolved under high pressure in DzO with photooxidation of some of the amino acids measured the 1.06pm continuous output of an Nd-YAG laser, in terms of oxygen uptake showed a marked depen- has been developed in this laboratory (Matheson et dence on pH. This was particularly evident in the al., 1974). The total quenching rate constants of phocase of histidine where the rate had a pK around tophysically generated singlet oxygen by amino acids 6.5, the same as for the imidazole dissociation. and proteins have been measured by observing their The interpretation of dye sensitized photooxidation competitive inhibition of the chemical reaction of in terms of singlet oxygen mechanism was first made bilirubin with singlet oxygen (Matheson et al., 1975). by Foote and Wexler (1964) who demonstrated a However, reliable estimation of absolute chemical similar reactivity for singlet oxygen generated chemi- reaction rates of the amino acids with singlet oxygen cally and by photosensitization. The singlet oxygen has been delayed by experimental difficulties, the mechanism, also called type I1 photosensitized oxi- most serious of which is estimation of the singlet dation (Schenk, 1957) proceeds by the photophysi- oxygen steady state concentration. This required cally generated triplet dye molecule forming singlet knowledge of the oxygen absorption cross-section at oxygen by energy transfer to ground state oxygen. 1.06pm in water, u, and the singlet oxygen lifetime Type I photosensitized oxidation involves hydrogen 7 , since the singlet oxygen steady state concentration or electron transfer between the triplet dye molecule is given by U T E [ O ~where ] ~ , E is the rate of irradiaand substrate, followed by reaction of the resulting tion of the sample and Oz the oxygen concentration. substrate radical with ground state oxygen. Dye In the preceeding paper (Matheson, 1979) gl'c and sensitized photooxidation can proceed via both Type a27 have been determined, where u1 represents the I and I1 mechanisms at the same time. This applies absorption cross section proportional to the first particularly to the photosensitized oxidation of amino power of the oxygen concentration and u2 that proacids, where the proportion of type I and type I1 reac- portional to the second power of oxygen concentions can vary with substrate concentration. The tration. problem of distinguishing between type I and I1 Amino acid chemical reaction rate constants with mechanisms has been discussed by Foote (1976). Sep- singlet oxygen differing somewhat from those of this aration of type I and I1 reaction for the dye sensitized paper have been reported previously (Matheson et al., photooxidation of amino acids has been made by 1976). These were subject to difficulties regarding the a79
I. B. C. MATHESON and JOHN LEE
880
steady state singlet oxygen concentration and experimental design. The present work represents completely new experimental data. We report here the chemical rate constants of reaction of amino acids with singlet oxygen. MATERIALS AND METHODS
The amino acids were supplied by CalBiochem (San Diego. CA) and the D 2 0 99.8% by Sigma Chem. Co. (St. Louis, MO). Solutions of amino acids were made u p in D,O and adjusted to the required pD by additions of small amounts of NaOD or DCI. pH's were meesured with a Fisher Accumet Model 140 meter with a glass electrode, and pD was taken as pH measured + 0.4 (Salomaa et al.. 1964). The salt concentration was less than mM for all experiments.The Nd-YAG laser and high pressure optical cell were as previously described (Matheson, 1979). It may be shown that the steady state concentration of singlet oxygen '02.is given by, where l/r 9 k,A: where E / N represents the number of photons per second at 1.06gm divided by Avogadro's number, and n is the optical cell cross-sectional area, 7.13 x 10-3dm2 for the cell used in the experiments. The results of the preceeding paper, Matheson (1978) gave u17 = 2.3 x lo-' sdm2 mole-' and U ~ T= 4.6 x 10-'sdm5 rnol-', and these kalues were used to analyze the experimental data. Given ['O,] then the chemical rate of reaction of an amino acid with singlet oxygen, k,, may be determined from the relation ( - d I n A ) / d t = k,['02]. Initial amino acid concentrations of 50pM or less were used for all experiments so that the contribution of amino acid physical quenching and chemical reaction to the singlet oxygen loss rate would be less than 20:4 of that due to the solvent D20. Amino acid concentrations, histidine methionine and alanine were measured by (Beckman Model 119C) amino acid analysis using standard procedures, and tryptophan, and tyrosine were measured fluorimetrically with a Turner Spectro 210 Fluorometer. Oxygen concentrations were estimated by means of Henry's Law (solubility of oxygen obtained from Handbook of Chemistry and Physics, 1969), and concentrations of 0.1-0.2 M at 20°C were used experimentally. RESULTS AND DISCUSSION
The four amino acids known to be most reactive towards photosensitized oxidation, histidine, trypto-
-'r
0
I
I
I
I
500
1000
1500
2000
t,
=
Figure 1. Loss of histidine by reaction with singlet oxygen as a function of time at pH 6.5 at 20°C. Initial histidine concentration 50 pM, oxygen concentration 0.195 M and E/N = 1.02 x IO-'s-' at 1.06pm used for all five experiments.
Table 1. Collected amino acid rate constants for chemical reaction with and physical quenching of singlet oxygen Photophysically generated '02 Amino acid His TrP Met TYr A la
k, 10
3 I .6 0.8 0.2
k,
+ k, 10 5. I
I .7 -
0.13
'02generated by * photosensitization k,
0.7 0.4 0.5 -
k , t k, 5 4 3 -
Photophysically generated singlet oxygen : all rate constants in units of M - ' s-' x lo-' k 30% at pD 8.4 and 20".
*Data of Nilsson et al. (1972) methylene blue photosensitization in H20/CH30H 1 : 1, pH unstated.
phan, methionine and tyrosine, and a representative other amino acid, alanine were chosen for initial study. The decay of one of these, histidine at p D 6.5, at 20°C is shown in Fig. 1. This exhibits first order decay and the scatter shown is typical of all the experimental data. The ground state oxygen concentration for these experiments was 0.195 M and over the )h timescale of the longest run the amino acid loss due to reaction with ground state oxygen was less than This was true for all the amino acids studied and thus reaction with ground state oxygen is negligibly small in comparison to that with singlet oxygen. This is true in the case of tryptophan where we had previously (Matheson et al., 1975) seen a first order decay rate with ground state oxygen of 1 0 - 3 s - ' whereas we presently see < 1 0 - 5 s - 1 . This is particularly strange in that the same high pressure cell was used 'for the early and recent experiments. The only explanation that can be offered is that the early experiments were carried out just after the cell had been constructed and machined and that the steel surface constituting the cell wall has subsequently changed due to repeated exposure to aqueous solutions. The aged wall surface is presumably much less effective in catalyzing the autooxidation. The rate constants for chemical reaction of photophysically generated singlet oxygen with the amino acids is shown in the first column of Table 1; the total rate constants for chemical reaction and physical quenching are given in the second column. The chemical reaction rates were calculated using the a1 and o2 values given above and the total quenching rate constants are the data of Matheson et al. (1975) recalculated using 7 = 3 5 p s (Matheson, 1979) rather than the 20 ps value of Merkel and Kearns (1972). In the case of histidine the exact agreement between the chemical and total rate constants is probably fortuitous. The statistical error for these experiments is typically 30%, and there is also a systematic error (due for example to using the wrong value for a7) of the same order so that the rates could differ by a factor
Reactions of '02 with amino acids
p=-.+-
'Or
-+ '0
+
4 0 Y 6 1 0
710
do
do
Id0
'lo
PD
Figure 2. Variation of reaction rate constant of histidine all for a starting concentration of 5 0 p M with singlet oxygen in D20 as a function of pD. Units M - l s - ' x lo-' at 20°C. fit for pK = 6.9. Depth of vertical bars represent standard deviation. of two or more and not be significantly different. Thus in the case of alanine where the 2 x lo6 M - ' s - ' chemical rate constant is apparently greater than the total rate constant of 1.3 x lo6 M - ' s - ' this difference is not significant. However, it does seem to be a safe generalization to say that the chemical reaction is the major part of the interaction of singlet oxygen with the amino acids studied as reported by Matheson rt al. (1976). The other two columns of Table 1 are the data of Nilsson et a/. (1972). the third column showing the chemical rate constants and the fourth the total rates. It is clear that the total interaction rates of Nilsson et al. are somewhat lower than the results of this work for photophysically generated singlet oxygen and their values for the chemical rate con-
XXI
stants are approximately an order of magnitude lower than the results of this work. How much of this difference is due to the mixed water/alcohol solvent used by Nilsson et al.'or due to the inherent difficulties in separating type I and.11 processes and chemical and physical interactions, is not clear. Figure 2 shows the rate constant for chemical reaction of photophysically generated singlet oxygen with histidine as function of pD. The results confirm the photosensitization results of Sluyterman (1962) and Weil (1965) which show that the rate of photooxidation follows the histidine pK curve. The present data also show that photooxidation of histidine units critical to enzymatic activity where the activity loss follows the histidine pK curve, could also be indicative of singlet oxygen reaction with histidine. An example of such a study is that of Westhead (1965) who inactivated enolase by Rose Bengal photosensitization. In summary the present results show that amino acids react rapidly with photophysically generated singlet oxygen, the interaction is largely chemical and that for histidine the reaction rate constant follows the histidine pK curve. This corrects our earlier conclusions, Matheson et al. (19751, which were that the total of chemical and physical quenching measured then represented largely physical quenching following Nilsson et al. (1972). Work is in progress to extend these studies to the inactivation of enzymes by photophysically generated singlet oxygen. Acknowledgements-This work was supported by NSF Grant CHE-76-10988 and we thank Mr. G. T. Herrington
for assistance.
REFERENCES
Foote, C. S. and S. Wtxler (1964) J. Am. Chem. Soc. 86, 388&3881. Foote, C. S. (1976) In Free Radicals iii Biology (Edited by W. A. Pryor). Vol. 11, pp. 85-133. Academic Press, New York. Handbook of Chemisrry and Physics (1969) 50th Ed. Chemical Rubber Publishing Co.. Cleveland. OH.
Matheson, I. B. C., N. U. Curry and J. Lee (1974) J . Am. Chem. SOC. 94, 3348-51. Matheson, I. B. C., R. D. Etheridge. N. R. Kratowich and J. Lee (1975) Photochem. Photohiol. 21. 165-1 71.
Matheson. I. B. C.. D. E. Brabham and J. Lee (1976) presented at VII International Congress on Photobiology. Rome, abstract p. 22. Matheson, I. B. C. (1979) Photochem. PhotobioI. 29. 875-878. Merkel, P. B. and D. R. Kearns (1972) . J . Am. Chem. SOC. 94, 7244-53. Nilsson, R., P. B. Merkel and D. R. Kearns (1972) Photochem. Photobiol. 16, 117-124. Salomaa. P., L. L. Schaleger and F. A. Long (1964) J. Am. Chem. SOC.86, 1-7. Schenk, G. 0. (1957) Agnew. Chem. 69, 579-599. Sluyterman, L. A. (1962) Biochim. Biophys. Acra 60, 557-561. Weil, L., W. G. Gordon and A. R. Buchert (1951) Arch. Biochem. Biophys. 33, 9&109. Weil. L. (1965) Arch. Biochem. Biophys. 110, 57-68. Westhead. E. W. (1965) Biochemisrry 4, 2139-2144. Westhead. E. W. (1972) Merh. Enzymol. (Edited by S. P. Colowick and N. 0. Kaplan), Vol. XXV. pp. 401-409. Academic Press, New York.
