FLUORESCENCE RESOLUTION OF THE INTRINSIC TRYPTOPHAN RESIDUES OF BOVINE PROTEIN TYROSYL PHOSPHATASE

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP), Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F, Fluorescence measurements show that Trp(39) is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp(49). Solute fluorescence quenching studies of W39F reveal that Trp(49) is, highly exposed to the aqueous environment, In contrast, Trp(39) is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion, The fluorescence contributions of Trp(39),and Trp(49) are additive, and their sum is equivalent to that observed for wild type BPTP, Calculated intramolecular distances between Trp(39) or Trp(49) and, 5-[[C(acetylamino)-ethyl]amino]naphthalene-1-sulfonate group covalently bound at Cys(12) or Cys(17) of the respective protein mutants, place Trp(49) within 10 Angstrom and Trp(39) at least 20 Angstrom from the active site, The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponential, The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting, Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP, These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of K-sv and k(q).