Reaction of thrombins with human antithrombin III. I. Enzyme activity losses unrelated to the inhibitory reaction and their prevention

Thrombosis Research
Z S Latallo, J A Hall

Abstract

Adsorption loss of thrombin may produce considerable systematic error when quantitative evaluation of its reaction with antithrombin III is based on measurements of changes in enzyme activity with time. At 25 degrees C, pH 7.8 (0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES) glass, siliconized glass, polypropylene and polystyrene surfaces adsorb thrombin in a similar manner. Its adsorption is a saturation reaction reaching an equilibrium after about three hours. At initial concentrations of enzyme convenient for assay with chromogenic substrate (10 nM or less) a large fraction of activity is lost. Binding of alpha thrombin to polypropylene is very tight. The association constants (Ka) were found to be 12.6 X 10(9) M-1 and 4.0 X 10(9) M-1 for human and bovine alpha thrombin respectively. The adsorbed enzyme has hardly any ability to hydrolyze chromogenic substrate. A small part of its activity can be recovered by displacement with PEG 6000, more when the substrate is present. The addition of non-ionic detergents and continuous stirring allows recovery of up to 30% of the enzyme activity and accelerates this process. Complete and long term (more than 20 hours at 25 degrees C) prevention of adsorption loss of human alpha and bovine alpha a...Continue Reading

References

Sep 1, 1975·Journal of Biomedical Materials Research·D F WaughH Ng
Sep 1, 1978·Journal of Biomedical Materials Research·D F WaughY R Freund
Jun 1, 1976·Thrombosis Research·W WasiewskiJ W Fenton
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Nov 1, 1983·Analytical Biochemistry·C H Suelter, M DeLuca
Feb 15, 1983·Biochimica Et Biophysica Acta·R Lottenberg, C M Jackson

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Citations

Aug 1, 1992·Protein Science : a Publication of the Protein Society·S D LewisJ A Shafer
Mar 17, 2019·Environmental Science and Pollution Research International·Xiaoxi ZhangZengwen Liu
Aug 8, 1997·The Journal of Biological Chemistry·P E BockI Björk
Jun 25, 2004·The Journal of Biological Chemistry·Paul D BoxrudPaul E Bock
Nov 6, 2015·Protein Science : a Publication of the Protein Society·Tihami Qureshi, Cynthia B Peterson

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