TIME-RESOLVED FLUORESCENCE AND ANISOTROPY OF COVALENTLY COUPLED 1-PYRENEBUTYRIC ACID FOR MONITORING THE CRYSTALLIZATION CONDITIONS OF LYSOZYME

Time-resolved fluorescence and anisotropy measurements of trace amount s of 1-pyrenebutyric acid labeled hen egg-white lysozyme (PBA-HEL) wer e used to characterize hen egg-white lysozyme (HEL) crystallization co nditions. The effects of sodium chloride and protein concentrations on the fluorescence lifetimes and rotational correlation times of the la beled protein were examined. These results were compared with the effe cts of the salts ammonium acetate and ammonium sulfate. Addition of pr otein precipitants caused increases in the rotational correlation time s which were attributed to a combination of steric, hydrodynamic, gene ral electrostatic and specific ionic interactions. This decrease in th e rotational mobility of HEL appears to be a necessary but not suffici ent condition to allow the formation of specific interactions leading to crystallization. The results demonstrated that fluorescence measure ments are effective in characterizing and monitoring protein crystalli zation processes prior to the appearance of macroscopic crystals.