Purification, Some Properties And Modification Of Groups Of The Thrombin From Oryctolagus Cuniculus

Thrombin (EC 3.4.21.5) is a natural enzyme in the body's Coagulation System, and it is also very important as a Serine protease. It can directly act on the fibrinogen, and hydrolyzes 4 Arg-Gly peptide bond of the fibrinogen, which makes fibrinogen become to fibrin for congealing the blood. Thrombin is widely used in pharmaceutical industry. It can be used in local bleeding, and also to treat diseases such as false aneurysm with fibrinogen together.Purified Thrombin solution from rabbit (Oryctolagus cuniculus) blood was prepared by barium citrate absorption first and further stepwise purified by dialysis, freeze-drying, DEAE-sepharose chromatography and Superdex S-200 gel filtration. The results showed purification times of 41.18, relative enzyme activity of 150.71U/mg, and recovery rate of 16.49%.The molecular weight of this enzyme is about 35KD. It is sensitive to pH and temperature and stable at pH between 5 to 8, with optimal pH at 7.4 and temperature at 38℃. Enzyme activity decreased rapidly at above 50℃. Enzyme activity could be activated by Zn²⁺,Cu²⁺ and largely inhibited by Fe²⁺. Also, inhibition effects were obviously observed in organic solvents with order: isoproponol＞ethylene glycol＞ethanol; and no significant inhibition effect was observed in methanol. Iodophors can strongly inhibit the activity of thrombin, and Glutaraldehyde and other exterior drugs have a certain inhibitory effect on it except Compound alcohol disinfectant. The Rabbit Thrombin can be used on medical industry by comparing its some properties with those of Human Thrombin.The thrombin was selectively modified by PMSF, NBS, IAc, BrAc, TNBS, SUAN, PCMB, DTT, NAI, BD, Chloramine-T and changes in the activities of the enzyme have been detected. The results showed that DTT is the most strongest inhibitor which inhabit the enzyme activities. PMSF, TNBS, SUAN, IAc, BrAc and Chloramine-T were also found to have inhibition effect on thrombin activity. NBS, BD and PCMB were found to have a little inhibition effect, and NAI has no effect. All of these suggested that Ser, His, Lysε-amino, Met sulfide-based and disulfide linkage should considered as indispensable groups of thrombin..