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. Human seminal plasma contains a variety of proteins secreted by seminal vesicles and the prostate. Human seminal plasma prostatic inhibin (HSPI) is one of first seminal plasma proteins that has been isolated, purified, and characterized. 1 It has 94 amino acids and has a molecular weight of 10.4 kDa. HSPI has been the subject of interest for more than a decade. Its biological activity ranges from preventing pregnancy to curing prostate cancer. Inhibin prevents pregnancy in mammals by modulating the level of circulating follicle stimulating hormones (FSH). 2 It suppresses prolactin, a hormone that promotes lactation. 3 Therefore, neutralizing inhibin through active immunization leads to increase in milk production. Seidah et al. have reported the primary sequence of HSPI. Materials and Methods Sample Preparation. HSPI has been isolated from the semen samples of healthy men obtained at infertility clinics and purified following the well-established procedure described elsewhere. Other chemicals used in this study were acrylamide (Sisco Research Laboratories Co., India), KI (potassium iodide, Qualigens Fine Chemicals Co., India), CsCl (cesium chloride, Spectrochem Pvt. Ltd., India), sodium thiosulfate (Na 2 S 2 O 3 , Qualigens Fine Chemicals Co., India), sodium acetate (Qualigens Fine Chemicals Co.), tryptophan (Sigma Chemical Co., United States), and NATA (N-acetyl tryptophanamide, Sigm