Quenching of the Tyrosyl and Tryptophyl Fluorescence of Subtilisins Carlsberg and Novo by Iodide

Michael F. Brown, Rodger A. Raubach, Thomas Schleich, Siraj Omar

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19 Scopus citations

Abstract

The tyrosyl and tryptophyl fluorescence of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively, is quenched efficiently by I- but is not significantly affected by Cs+. The I- quenching data were analyzed using a modified Stern-Volmer treatment (Lehrer, S. S. (1971), Biochemistry 10, 3254), yielding values for the effective fraction of accessible protein fluorescence of 90-95 and 88-92% for the tyrosyl and tryptophyl emission of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively. Similar values were obtained at pH 5 and 7. The effective collisional quenching constant depends on pH in a manner suggesting the participation of protein surface charge in the quenching mechanism. Significant singlet energy transfer (efficiency = 0.52) from tyrosyl to tryptophyl residues was inferred from the excitation spectra of diisopropylphosphorylsubtilisin Novo. The very low efficiency of energy transfer to Trp-113 in diisopropylphosphorylsubtilisin Carlsberg suggests that Trp-105 and Trp-241 are the acceptors of tyrosyl emission in the homologous Novo enzyme. The unusually low quantum yield of Trp-113 in diisopropylphosphorylsubtilisin Carlsberg together with the tryptophyl fluorescence quenching behavior of the Novo enzyme suggests that this residue is “buried” and inaccessible to quenching in both enzymes. The tyrosyl quenching behavior of diisopropylphosphorylsubtilisin Carlsberg is consistent with the high degree of solvent exposure of aromatic residues evident in the x-ray model of subtilisin Novo.

Original languageEnglish (US)
Pages (from-to)987-992
Number of pages6
JournalBiochemistry
Volume16
Issue number5
DOIs
StatePublished - Mar 1 1977
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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