Tyrosine and tyrosinate fluorescence of bovine testes calmodulin: calcium and pH dependence.
At physiological pH, bovine testes calmodulin (t-CaM) upon excitation at 278 nm shows typical tyrosine fluorescence at 305 nm and a spectral band characteristic of emission from tyrosinate , at 330-350 nm. In addition, a new band at 312-320 nm appears upon excitation at 288 nm. The pH dependence of the excitation spectra demonstrates that the intense tyrosinate emission at 330-355 nm originates from direct excitation of ground-state tyrosinate . The tyrosinate emission shows complex pH dependence and reaches its highest intensities at pH 7.0 and 8.5, in both apo (Ca free) and holo (Ca saturated) t-CaM. The evidence suggests that the major contribution to the tyrosinate emission at 330-350 nm originates from Tyr-99. In holo t-CaM, the tyrosine emission at 305 nm is quenched at basic pH values and exhibits a sigmoidal pH titration curve with pK(app) 7.0. The tyrosine emission in apo t-CaM is weaker and is almost insensitive to changes in pH. The pH dependence of the emission at 316 nm is the same as the pH dependence of the tyrosine emission in both apo and holo t-CaM. The differences between the fluorescence of apo and holo t-CaM are attributed to a Ca2+-induced shift in the pKa of carboxylic side chains located in the immediate vicinity of the tyrosine residues. The enhancement of the fluorescence by Ca2+ is pH dependent and is maximal at pH 6.5. Above pH 8.0, there is almost no Ca2+ effect on the fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008297:Male; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013737:Testis; D014443:Tyrosine
D002147:Calmodulin; D014443:Tyrosine; D002118:Calcium