FLUORESCENCE ANALYSIS OF PH-INDUCED CONFORMATIONAL-CHANGES IN SPERM WHALE APOMYOGLOBIN .2. MECHANISM OF QUENCHING AT NEUTRAL PH, IDENTIFICATION OF QUENCHING GROUPS, AND CONFORMATIONAL PROPERTIES OF APOMYOGLOBIN

The fluorescence lifetimes of intact sperm whale apomyoglobin and HNB-apomyoglobin, produced by modification with Koshland's reagent at Trp-7, were studied as a function of pH. The fluorescence of apomyoglobin derivatives modified with iodoacetamide, bromoacetate, and methylisothiocyanate at histidine residues and at the N-terminus alpha-amino group was also studied, in the pH range 2-12.5. A comparison of the pH-dependent fluorescence of intact and modified apomyoglobins showed that the reduction in fluorescence intensity of apomyoglobin on reduction of from 8.5 to 5.5 resulted from quenching of emission by Trp-14 by a charged His residue, which is ionized over this pH range. This residue is probably His-119 (GH1), though it is possible that the "internal" His-24 (B5) may participate in quenching. The quenching mechanisms appear to be dynamic as the lifetime and intensity of fluorescence were proportional, which was especially clear in the case of HNB-apomyoglobin, where only Trp-14 fluoresced. Analysis of the Trp fluorescence of intact and modified apomyoglobins showed differences between the apo- and holoproteins in the conformations of the N-terminal and adjacent regions of the structure, and allowed the pH-dependent changes in the conformation of these regions to be followed. A relationship between the conformational states of the heme cavity and the N-terminus of apomyoglobin is suggested.