CONTRIBUTION OF INDIVIDUAL TRYPTOPHAN RESIDUES TO THE FLUORESCENCE-SPECTRUM OF NATIVE AND DENATURED FORMS OF HUMAN CARBONIC-ANHYDRASE-II

Measurements were made of fluorescence spectra produced by pseudo-wild -type human carbonic anhydrase LT and mutants in which the tryptophan residues had been replaced by phenylalanine or cysteine residues. 2D N MR spectra of N-15-labeled proteins indicated that the mutations had e ssentially no long range effects on structure and that the pertubation s of structure in the vicinity of the mutated Trp were small. The indi vidual contributions of the seven tryptophan residues were deduced fro m measurements on native proteins and on proteins subjected to various denaturing conditions. Trp97 and Trp245 are the major fluorescence em itters in the native state, contributing 52% and 38%, respectively, to the total fluorescence intensity. Comparisons of the fluorescence yie ld of pseudo-wild-type human carbonic anhydrase II and mutant proteins also indicate net energy transfer from Trp16 to Trp5 and from Trp192 to Trp209. The fluorescence from Trp5 is efficiently quenched by His64 . In addition, acrylamide quenching of fluorescence was used to probe the environment of tryptophans in proteins incubated in 0, 1.5, and 5 M guanidine hydrochloride. The results indicate that the part of the n ative protein that corresponds to beta-strands 3-7 forms a compact cor e in a molten globule intermediate.