Rate constants studies of the dye-sensitized photoinactivation of lysozyme

Abstract

The rate constants for the photodynamic inactivation of hen egg-white lysozyme at different temperatures were studied. Arrhenius plots of the methylene blue sensitized photo-inactivation of lysozyme gave an experimental activation energy of 7.5 kcal/mol. The rate constants for the photodynamic inactivation of lysozyme in the presence of riboflavin decreased almost linearly in the temperature range 4-38 degrees C. The photosensitized oxidation of lysozyme at -20 degrees C in freezing and non-freezing solvents was possible only in the presence of riboflavin. The effect of dye concentration on the quantum yield and rate constant for the photodynamic inactivation of lysozyme was examined. The quantum yields were lower when the concentrations of methylene blue used were low, and increased on increasing dye concentration, getting to a maximum and then declined at higher dye concentrations. It was found that in the case of riboflavin sensitized photo-inactivation of lysozyme both the rate constant and the quantum yield increased as the dye concentration increased. No maximum was observed over the range of dye concentrations studied. A new mechanism is postulated for the photodynamic action of lysozyme in the presence of riboflavin.

Isolation and photo-oxidation of lysozyme fragments

“…The activity of the enzyme was assayed by measuring the lysis of Micrococcus luteus (formerly named M. lysodeikticus) (Silva 1979;Shugar 1952).…”

“…Photo-oxidation of lysozyme dye-sensitized by riboflavin has been studied in detail (Risi et al 1973;Silva et al 1974Silva et al , 1977Silva 1979), and can be summarized as follows: His and Trp residues present in the enzyme are selectively photo-oxidized. The only His residue is destroyed according to a very well defined first-order kinetic, whereas the destruction of the six Trp residues shows an hyperbolical pathway in a semi-logarithmic plot.…”

Binding of Riboflavin to Lysozyme Promoted by Peroxidase‐generated Triplet Acetone

“…Amino acid analyses were carried out in a Beckman 120C amino acid analyzer by the method of Matsubara and Sasaki (1969). Lysozyme activity was measured by the method of Silva (1979).…”

“…H H potential for flavin photochemistry in the dark. An interesting possibility is the binding of flavins to macromolecules (Risi et al, 1973;Silva et al, 1974;Silva and Gaule, 1977;Silva, 1979). The photobinding of riboflavin to other molecules has been described by Oster and Wasserman (1962), Kostenbauder and De Luca (1963) and McKnight and Spikes (1979a,b).…”

Hemolysis of Human Red Blood Cells by Riboflavin-Cu(II) System: Enhancement by Azide

“…Several reports have shown that photoactivated riboflavin reacts with molecular oxygen to generate reactive oxygen species (ROS) [9,10]. These ROS have been shown to damage rat lens [11], inactivate enzymes [12,13], cause lipid peroxidation [14], increase protein cross linking [15], and destroy bilirubin [16], uric acid [17], and amino acids [18,19]. The damage caused by ROS is further exacerbated if the antioxidant enzymes themselves are damaged and inactivated by such events [20].…”