Study of prothrombin activation by taipan snake venom using light beating spectroscopy
Agarwal, G. P. (Gopal Prasad)
Armeniades, C. D.
Master of Arts
In this study, the recently developed technique of light beating spectroscopy has been used for the first time to measure the dynamic properties of prothrombin and its activation kinetics via Taipan Snake Venom, micellar phospholipid and Ca++. The correlation times fCc) on the samples of human prothrombin, which were prepared by membrance filtering techniques, were measured as a function of scattering angle to determine the particle size uniformity. P vs K plot was found to be linear for one sample at 1°C, but nonlinearity was seen for other two samples of human prothrombin at 2°C, indicating some aggregation. The translational diffusion coefficient was measured on more than 2 samples at 2 mg/ml. The average value of diffusion coefficient (D2,Tvf ) was measured to be 4. 72 x 1 -7c m2/sec with a standard deviation of . 23 x 1"-7c m2/sec. The diffusion coefficient value for a sample of 5 mg/ml human prothrombin was 4. 51 x 1"-7c m7^/sec. These values are in agreement with the reported values of 4. 8 x 1“-c7m / sec2and 4-. 67x 1 2cm /sec at the respective concentrations. Sedimentation coefficients determined from sedimentation velocity experiments were 5, 37S and 6. 7S at 2 mg/ml and 5 mg /ml. These were higher than the reported values of 4. 8S and 5. 2S for the corresponding concentrations, which could be attributed to aggregation at very low ionic strength and low pH. The molecular weights of human prothrombin calculated from sedimentation and diffusion coefficients data were found to be 92, and 12, at 2 mg/ml and 5 mg/ml concentrations, respectively. From a sedimentation equilibrium experiment on a sample of first lot human prothrombin diluted to . 2 mg/ml, the sample was found to be homogeneous species having 69, molecular weight. In solution, the conversion of prothrombin was observed by monitoring the change of translational diffusion coefficient during its activation process. A single clipped autocorrelation function at low counts per sample interval (<<1) and clipping level k = , is approximately equal to .<I(t) I(t+T)> . Consequently, the autocorrelation function is proportional to the second power of M and c and therefore very sensitive to the presence of big particles in the solution. To satisfy this requirement nonphysiological activator Taipan Snake Venom (oxyuranus Scutellatus) and micellar phospholipid, Dihexanoyllecithin were used which gave negligible contribution to the autocorrelation function. An unusual y long time of 3-5 days was observed for the complete conversion of first lot of human prothrombin. This slow rate could be due to possible conversion of prothrombin to Intermediate I and inhibitory fragment 1 prior to the activation reaction. These products retard the activation rate by an order of magnitude. Other factors such as the low surface area per unit mass of the micellar phospholipid (compared to the commonly used bilayer phospholipid), the low concentration of CaCl (.3mM) set by its precipitation threshold and the use of low temperatures to avoid aggregation may also contribute to the slow conversion. For a sample of second lot human prothrombin 18% change in diffusion coefficient was observed at 2°C in less than an hour. Complete conversion study could not be done as the solution aggregated. This supports our hypothesis that a prior conversion of prothrombin of first lot had taken place.