Fluorescence Quenching of Aminopeptidase‐I from Pronase

摘要

AbstractAminopeptidase‐I from pronase contains five tryptophan residues, determined by modification withN‐bromosuccinimide and MCD measurments. The fluorescence of the enzyme is quenched by acrylamide, a neutral quencher, but not by iodide, an anionic quencher. The quenching constant for acrylamide is about one tenth that of acctyltryptophan in aqueous solution because of the steric effect of the large protein molecule. The inaccessibility of iodide to the tryptophans is probably due to the anionic environment of the fluorophores created by the large content of Asp and Glu in the enzyme. Aminopeptidase‐I is quenched by Cu(II) and Co(II) through the formation of a nonfluorescent ground‐state complex. The results indicate that there are three accessible tryptophans for Cu(II) and two for Co(II) suggesting that some tryptophans are buried inside the enzyme and are thus inaccessible to the quencher. In the presence of a competitive inhibitor, the enzyme contains one fewer accessible tryptophan for Cu(II) indicating that this tryptophan is located in the active center. The CD spectrum of the enzyme in the far UV region is unaltered by the presence of Ca(II), whereas that in the aromatic region is enhanced significantly. The perturbation of the CD spectrum probably results from the local conformational change induced by Ca(II) or from the rigidity of the tryptophan being enhanced by the bound