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Abstract— Electron‐scavenging experiments with N2O as scavenger demonstrate at least two electron‐producing reactions of the excited singlet states of the exciplex species formed by indole or 1 ‐methyl‐indole with water. Most electrons reacting with N2O result from collision of the scavenger with a metastable state formed from the initial exciplex state but finite electron yields from indole and 1‐methyl‐indole at limiting scavenger concentrations suggest that the intermediate states also eject electrons directly into the solvent. The formation of the first metastable state from the fluorescent exciplex state has an activation energy, EM, estimated to be about 13 kcal/mole for both indole and 1 ‐methyl‐indole water exciplexes. The EM values for 1‐methyl‐indole from fluorescence and electron yields are the same, Indicating that at neutral and alkaline pH fluorescence quenching and electron extraction are both being controlled by the formation of the first metastable intermediate. Observed electron yields from indole‐water and indole‐methanol exciplexes are less than predicted using fluorescence data, although EM values of 1 kcal/mole are obtained for the indole‐methanol exciplex by both methods. At pH 12·0 and 28°C the total electron yields for indole‐water and 1 ‐methyl‐indole‐water exciplexes are 0·30 and 0·25, respectively. The residual yields attributed to outright formation of hydrated electrons from the initial exciplex excited stateare 0·11 and 0·05, respectively. Electron yields from the indole‐water exciplex are strongly pH dependent only near pH 1 where the fluorescence yields as well as the electron yields decrease rapidly with increasing acidity. The 1‐methyl‐indole‐water exciplex shows an additional pH dependence which is first‐order in hydrogen‐ion activity and has an effective pKa of about 11·5. Comparable yields for indole and 1‐methyl‐indole are found only above pH 12. High electron yields are found with indole in the exciplex‐forming solvent dioxane and in the non‐exciplex forming solvent cyclohexane. For the latter system electrons are probably derived only from the lowest excited state of indole on collision with N2O.

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Egg white lysozyme was inactivated by photodynamic treatment in sodium phosphate buffer at pH8 using methylene blue, eosin Y and FMN as sensitizers. Measurements sensitive to changes in protein conformation, in particular, tryptophyl fluorescence and protease digestibility, were made during the course of inactivation. The rate of change of lysozyme tertiary structure as measured in these ways correlated closely with the rate of loss of enzyme activity during photodynamic treatment. Further, forms of lysozyme which were enzymatically active, but which were more sensitive to high temperature than native enzyme were produced by photodynamic treatment. It is concluded that the photodynamic inactivation of lysozyme under the conditions used results largely from the photooxidation of amino acid residues essential for the maintenance of the catalytically active conformation of the enzyme.INTROD UCTl ON ENZYMES exposed to visible light in the presence of oxygen and a sensitizing dye lose catalytic activity due to the photooxidation of susceptible amino acid residues including histidine, tryptophan, tyrosine, methionine and cysteine [ 11. Such dye-sensitized photooxidation reactions are commonly termed 'photodynamic action" I]. I n contrast to ultraviolet irradiation, peptide bonds and disulfide bonds in proteins are not broken by photodynamic treatment [2,3].Those amino acid residues involved in the maintenance of enzymic activity have been identified by correlating their modification with loss of enzymic activity [4,5]. Photodynamic treatment of enzymes has been utilized extensively for such studies [6-81. The loss of enzymic activity during photodynamic treatment can occur, not only by the chemical alteration of amino acid side chains located in the active site of the enzyme, but also by the destruction of amino acid residues essential for the maintenance of the native conformation. This latter mechanism has not been considered in most studies of the photodynamic inactivation of enzymes, and in those cases where conformational measurements have been made, the biophysical techniques used often lacked the sensitivity necessary to detect small but important changes in enzyme conformation.Lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17, hen's egg white) was selected for the present study since previous investigations indicated that conformational changes occurred during its photodynamic inactivation . Weil et al.[9], found some years ago that the solubility of the enzyme decreased while its relative viscosity increased during photodynamic inactivation. In more recent studies, photooxidized lysozyme showed an increased susceptibility to tryptic hydrolysis [ 10-1 21, an increased levorotation [ 10-121, changes in ultraviolet absorption [ 10-121 and a decreased thermodenaturation midpoint [ I 1 , 121.