This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.106.088740v1 91/10/3864    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zelent, B. Articles by Gryczynski, Z. Search for Related Content PubMed PubMed Citation Articles by Zelent, B. Articles by Gryczynski, Z.

Protonation of Excited State Pyrene-1-Carboxylate by Phosphate and Organic Acids in Aqueous Solution Studied by Fluorescence Spectroscopy

Bogumil Zelent ^*, Jane M. Vanderkooi ^*, Ryan G. Coleman ^*, Ignacy Gryczynski  and Zygmunt Gryczynski 

^* Department of Biochemistry & Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and Department of Cell Biology and Genetics, University of North Texas, Health Science Center, Fort Worth, Texas

Correspondence: Address reprint requests to Jane M. Vanderkooi, E-mail: vanderko{at}mail.med.upenn.edu.

Pyrene-1-carboxylic acid has a pK of 4.0 in the ground state and 8.1 in the singlet electronic excited state. In the pH range of physiological interest (pH 5–8), the ground state compound is largely ionized as pyrene-1-carboxylate, but protonation of the excited state molecule occurs when a proton donor reacts with the carboxylate during the excited state lifetime of the fluorophore. Both forms of the pyrene derivatives are fluorescent, and in this work the protonation reaction was measured by monitoring steady-state and time-resolved fluorescence. The rate of protonation of pyrene-COO^– by acetic, chloroacetic, lactic, and cacodylic acids is a function of pK, as predicted by Marcus theory. The rate of proton transfer from these acids saturates at high concentration, as expected for the existence of an encounter complex. Trihydrogen-phosphate is a much better proton donor than dihydrogen- and monohydrogen-phosphate, as can be seen by the pH dependence. The proton-donating ability of phosphate does not saturate at high concentrations, but increases with increasing phosphate concentration. We suggest that enhanced rate of proton transfer at high phosphate concentrations may be due to the dual proton donating and accepting nature of phosphate, in analogy to the Grotthuss mechanism for proton transfer in water. It is suggested that in molecular structures containing multiple phosphates, such as membrane surfaces and DNA, proton transfer rates will be enhanced by this mechanism.