A comparative kinetic study on the singlet molecular oxygen-mediated photoxidation of alpha- and beta-chymotrypsins

Kinetic aspects of the sensitized photooxidation of alpha- and beta-chymotrypsins have been studied at pH 6 and 8. The sensitization, employing classical O-2((1)Delta(g))-photogenerators, such as xanthene dyes, is a kinetically intricate process because of the presence of ground state dye-protein associations and to the simultaneous participation of superoxide ion and singlet molecular oxygen [O-2((1)Delta(g))]. Both proteins, that possess the same distribution pattern of photooxidizable amino acids, suffer a pure O-2((1)Delta(g))-mediated photodynamic attack, using the carbonylic sensitizer Perinaphthenone. Overall and reactive rate constants for the O-2((1)Delta(g))-quenching (in the order of 10(8) and 10(7)/M/s, respectively), and rates of oxygen consumption determined by time-resolved, spectroscopic and polarographic methods indicate that alpha- and beta-chymotrypsins are less photooxidizable at pH 6, as a result of an enhancement of the O-2((1)Delta(g))-physical quenching component. In general terms, beta-chymotrypsin exhibits the greater overall proclivity to interact with O-2((1)Delta(g)), whereas structural factors, possibly evidenced by a higher exposure of the reactive tryptophan residues, impart an increased photooxidation degree to the proteins at pH 8, specially to the alpha-chymotrypsin.