INVESTIGATION OF ELECTRONIC AND STERIC ENVIRONMENTS OF TYROSYL RESIDUES IN RIBONUCLEASE A AND ERWINIA-CAROTOVORA L-ASPARAGINASE THROUGH FLUORESCENCE QUENCHING BY CESIUM, IODIDE AND PHOSPHATE IONS

The fluorescence lifetimes and relative quantum yields of several derivatives of tyrosine are reported. The fluorescence quenching of these compounds by .**GRAPHIC**. Cs+ and I- was investigated. The encounter rate constants calculated from the quenching parameters and lifetimes, show a clear dependence on the charges born by quenchers and fluorophores. The ratio of the Stern-Volmer constants of I- and Cs+ ions of similar size defines an electrostatic parameter sensitive to the charge of the fluorophore which can be evaluated without knowledge of the fluorescence lifetimes. The mean of the encounter rate constants for Cs+ and I- defines a rate constant which is largely charge-independent and is used to establish a steric parameter. The 2 parameters are used to investigate the tyrosine environment in bovine RNase A (EC 3.1.4.23) and E. carotovora L-asparaginase (EC 3.5.1.1). The quantum yield of L-asparaginase (0.12) is very high for a class A protein and may be associated with the absence of disulfide bridges. There was no evidence for more than 1 type of tyrosine residue from the quenching experiments with either enzyme, an observation which is attributed to efficient energy transfer among tyrosine residues. At pH values close to the isoelectric points of the enzymes the electrostatic parameter suggests that the environment of the quenchable tyrosines in L-asparaginase is more positive than in RNase [EC 3.1.4.23]. in 1% sodium dodecyl sulfate the tyrosine environment of L-asparaginase becomes negative. The steric parameter indicates a lower accessibility of the tyrosine residues in L-asparaginase than in RNase [EC 3.1.4.23]. An illustrative calculation is provided linking the steric parameter with the number of exposed tyrosine residues, by considering the greater collision frequency of the larger protein molecules and the encounter distance for quenching determined from charge effects on the quenching of the model compounds. The calculation indicate that 3 tyrosyl residues are accessible in RNase. In L-asparaginase the number increases from 0.4 at pH 5.73 to 0.8 at pH 9.16 suggesting a loosening of the enzyme structure at high pH.