Fluorescence and NMR studies of human seminal plasma prostatic inhibin: association of lifetimes with sterically constrained tryptophans

Abstract

Human seminal plasma prostatic inhibin (HSPI) is one of the first seminal plasma proteins, which has been isolated, purified, and characterized. HSPI contains two tryptophans at positions 32 and 92 along its 94 amino acid primary sequence. Among the three fluorescence quenchers acrylamide (neutral), potassium iodide (anionic), and cesium chloride (cationic), potassium iodide is found to quench the tryptophan fluorescence more compared to the other two. The fluorescence decay of HSPI is biexponential with lifetimes 5.86 and 2.44 ns. The Stern-Volmer quenching curves in the case of fluorescence intensity and average lifetime are identical, indicating that the quenching mechanism is purely dynamic. The decay associated spectra of the two lifetimes show that the two tryptophans are solvent-exposed. The fluorescence quenching data is in favor of associating the two lifetimes to separate tryptophans. The fluorescence anisotropy decay of HSPI is single exponential with a correlation time of 9.2 ns, which is due to the rotation of entire protein. Absence of any fast component in the anisotropy decay indicates that the two tryptophans are in motionally restricted, rigid environments. In NMR studies, the cross-peak patterns observed in 2D-COSY and 2D-NOESY spectra of HSPI gave unambiguous evidence that each of the two tryptophans is sterically constrained and exists in a single rotamer population.