Thrombin Applications
Production of fibrin clot in plasma:
Typicallty one to two units of Thrombin will clot one mL of plasma.
Cleavage of Fusion Proteins:
Thrombin can be used for the cleavage of many peptides at the Thrombin recognition site using concentrations of 0.5 NIH units thrombin per one nanomole polypeptide in 20 microliters of 50 mM ammonium bicarbonate, pH 8.0.
Thrombin cleavage of fusion proteins can be carried out at a Thrombin to fusion protein ratio of 1:500.
Fusion proteins may be cleaved in Thrombin cleavage buffer consisting of 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 and 0.1% 2-mercaptoethanol. 2 mg of fusion protein was incubated with 4 µg of thrombin for 20 minutes at RT in the cleavage buffer.
Several conventions are used in Thrombin literature:
1 IOWA unit= 0.83 NIH unit
1 WHO unit = 0.56 NIH unit
1 NIH unit = 0.324 +/- 0.073 µg
1 NIH unit = 1 USP unit
Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-arg-nitrobenzyl ester, and a thiobenzyl ester synthetic substrates
thrombin references
1.Enzyme Nomenclature: EC 3.4.21.5
2.Chang, J.Y., Eur. J. Biochem., 151, 217?224 (1985).
3.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed, p. 148.
4.Machovich, R., The Thrombin, 1, 63-66 (1984)
5.Machovich, R., The Thrombin, 1, 111 (1984)
6.Prasad, S., J. Biol. Chem. 279, 10103-10108 (2004)
7.Kisiel, W., Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J. Clin. Invest. 64, 761-769, (1979)
8.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed: Table 2. See also: The Enzyme Explorer: Plasma and Blood Protein Resource
9.Qian, W.J., et al., J. Proteome Res., 4, 2070-2080 (2005).
10.Nilsson, B., et al., Arch. Biochem. Biophys., 224, 127-133 (1983)
11.Boyer, P.D., The Enzymes, Academic Press (New York), 3rd ed., Vol. III, p. 277-321 (1971).
12.Expasy/SwissProt: P00743
13.Boissel, J.P., et al., J. Biol. Chem., 259, 5691-5697 1984).
14.Righetti, P.G., and Tudor, G., Isoelectric points and molecular weights of proteins, a new table. Journal of Chromatography, 220, 115-194 (1981).
15.Butkowski, R.J. et al., J. Biol. Chem., 252, 4942 (1977).
16.Winzor, D. J. and Scheraga, H. A., Arch. Biochem. Biophys. 104, 202-207 (1964)
17.Human Blood Coagulation, Haemostasis and Thrombosis, 2nd ed., R. Biggs, ed., p. 722 (1976).
18.The Handbook of Synthetic Substrates, Hemker, H. C., Martinus Nijhoff publisher (1983).
19.Lottenberg, R., et al., Assay of Coagulation Proteases Using Peptide Chromogenic and Fluorogenic Substrates. Meth. Enzymol., 80-C, 341-361 (1981).
20.Chang, Y., Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem., 151(2), 217-224 (1985).
21.Hakes, D.J. and Dixon, J.E., Anal. Biochem., 202, 293 (1992).
22.Gaun, KL and Dixon, JE,, Anal. Biochem., 192, 262, 1991
23.De Cristofaro, R. and De Candia, E., J. Thromb. Thrombolysis, 15, 151-163 (2003)
24.Sherwood, J.A., Mol. Biochem. Parisitol., 40, 173-181 (1990)
25.Berg, D.T., et al., Science, 273, 1389-1391 (1996)
26.Lundblad, R.L. et al., Methods Enzymol., 45, 156 (1976)
27.Matsuoka, S., et al., JP. J. Pharmacol., 51, 455-463 (1989)
28.Wimen, B., Meth. Enzymol., 80, 395-408 (1981)
29.Magnusson, S. The Enzymes, 3rd ed., III, pp. 277-321, Boyer, P.D., ed., Academic Press (1971)
