| Abstract: | A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge. |
