CHARACTERIZATION OF PYRIDOXAL-PHOSPHATE AS AN OPTICAL LABEL FOR MEASURING ELECTROSTATIC POTENTIALS IN PROTEINS

This paper presents data which allow one to characterize pyridoxal 5'- phosphate (PLP) as an optical label for electrostatic potential measur ements in proteins. Experimental studies were carried out with 6-N-(5' -phosphopyridoxyl)-2-N-acetyl-lysyl methyl ester (PLP-ALME) as a model compound which simulates PLP covalently bound to a protein. Calculati ons of electrostatic potential maps were done using the model compound 2,4-dimethyl-3-hydroxy-5-hydroxymethyl phosphate-pyridine (DHHPP). St udies on relative changes in PLP-ALME fluorescence and absorbance vs. pH resulted in four pK(a) values of PLP-ALME in solution which can be used as intrinsic pK(a) values (pK(int)) of the ionizable groups of th e label covalently bound to the protein. The pK(a) values obtained fro m fluorescence data are 4.1, 5.6, 8.7 and 11.1 and those from absorban ce data are 3.1, 4.7, 8.7 and 11.0. The differences between correspond ing pK, values are related to differences in PLP-ALME behaviour betwee n the excited and ground electronic states and to intramolecular charg e-charge interactions. Quenching of PLP-ALME fluorescence by I-, acryl amide and Cs+ at pH 6, 7 and 8 shows that in the case of I- and acryla mide the Stem-Volmer constants of quenching (K-SV) decrease with incre asing pH, while the opposite is true for K-SV of Cs+. These results as well as the analysis of electrostatic potential maps of DHHPP show th at with its ampholytic character PLP can be used to measure local elec trostatic potentials in the pH range 5-9.