S. K. Ravi

Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma

Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigated in the native state, in the presence of PrC and phosphatidylcholines (PCs) with short (valeryl, C-5) and long (myristoyl, C-14) chains, and upon denaturation using fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) measurements. The results obtained show that the environment of tryptophan residues in HSP-1/2 is more heterogeneous as compared to that in PDC-109. Binding of choline containing ligands afforded a protection to the tryptophan residues with the shielding order being: PrC≤divalaroyl PC<dimyristoyl PC. REES value obtained for HSP-1/2 (3.5nm) is smaller than that of observed for PDC-109 (4nm) and binding to PrC and DVPC reduced the REES to 1nm. HSP-1/2 exhibits only partial unfolding with chemical denaturants with no cooperativity, whereas complete unfolding was observed in the presence of 10mM dithiothreitol, indicating that disulfide linkages prevent complete unfolding of the protein. In the presence of PrC the transition midpoints shifted to higher concentrations of the denaturant together with a broadening of the sigmoidal transitions, indicating that ligand binding as well as polydispersity modulate the unfolding process.

Effect of Oral Supplementation of Vitamin C and Exercise on Plasma Vitamin C Status in Marwari Horses

The objective of the present study was to evaluate the effect of the oral supplementation with ascorbic acid on plasma levels in Marwari horses. The experiment was planned to supplement ascorbic acid @ 5 mg/kg. Body weight orally for 4 days on plasma ascorbic acid levels in horses. There was no significant difference in plasma ascorbic acid levels between day 1st and day 5th in supplemented group and control group. After that, a second trial was undertaken with supplementation of sodium ascorbate @ 20 mg/Kg body weight for 30 days in exercising horses. Exercising horses were under gallop + canter exercise of 5 km per day in sandy track. Blood samples were taken on each 10th day of the study period. There was no significant difference found in plasma ascorbic acid levels between supplemented and non-supplemented group of exercising horses. It was also found that plasma ascorbic acid levels were also significantly low in exercising non-supplemented horses than the horses which were under rest. So it was concluded that continuous 5 km gallop+ canter exercise in sandy track for a month reduces plasma ascorbic acid levels in horses but it cannot be replenished by oral supplementation of ascorbic acid because bioavailability of ascorbic acid on oral supplementation in Marwari horses is very poor.