An investigation of the electronic and steric environments of tyrosyl residues in ribonuclease A and Erwinia carotovora L-asparaginase through fluorescence quenching by caesium, iodide and phosphate ions.

The fluorescence lifetimes and relative quantum yields of several derivatives of tyrosine are reported. The quenching of the fluorescence of these compounds by phosphate, caesium and iodide ions has been investigated; the encounter rate constants, calculated from the quenching parameters and lifetimes, show a clear dependence on the charges borne by the quenchers and fluorophores. The ratio of the Stern-Volmer constants of iodide and caesium, ions of similar size, defines an electrostatic parameter sensitive to the charge of the fluorophore which can be evaluated without knowledge of the fluorescent lifetimes. The mean of the encounter rate constants for caesium and iodide ions defines a rate constant which is largely charge-independent and is used to establish a steric parameter. The two parameters are used to investigate the tyrosine environment in bovine ribonuclease A (EC 3.1.4.23) and Erwinia 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 disulphide bridges. There was no evidence for more than one type of tyrosine residue from the quenching experiments with either enzyme, an observation which is attributed to efficient energy transfer amongst 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 somewhat more positive than in ribonuclease. In 1% sodium dodecyl sulphate the tyrosine environment of L-asparaginase becomes markedly negative as expected. The steric parameter indicates a lower accessibility of the tyrosine residues in L-asparaginase than in ribonuclease; an illustrative calculation is provided linking the steric parameter with the number of exposed tyrosine residues by taking into account 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 suggests that three tyrosyl residues are accessible in ribonuclease, in good agreement with other studies, but 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.