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The structures and proteolytic specificities of autolysed human thrombin
The Biochemical Journal
|December 15, 1986
Summary
Human alpha-thrombin undergoes autolysis at specific Arg/Lys bonds, revealing new intermediates like beta-prime-thrombin and demonstrating comparable activity of autolysed forms. This study quantifies autolytic sites and their impact on thrombin structure and function.
Area of Science:
- Biochemistry
- Enzymology
- Proteolysis
Background:
- Human alpha-thrombin is a key enzyme in coagulation.
- Autolysis, or self-degradation, is a known process affecting enzyme stability.
- Understanding thrombin autolysis is crucial for its biological role and therapeutic applications.
Purpose of the Study:
- To identify and quantify the major autolytic sites in human alpha-thrombin.
- To investigate the intermediates formed during thrombin autolysis.
- To compare the proteolytic activity of native and autolysed thrombin forms.
Main Methods:
- Incubation of alpha-thrombin under controlled conditions (50 mM ammonium bicarbonate, 25°C).
- Purification and analysis of generated peptide fragments.
- High-performance liquid chromatography (HPLC) for quantitative analysis of thrombin forms.
Main Results:
- Identified three major autolytic sites: Lys-Gly (154-155), Arg-Tyr (70-71), and Arg-Asn (73-74) with 50% cleavage times of 32h, 72h, and 96h, respectively.
- Discovered a novel intermediate, beta'-thrombin (cleavage at Lys-Gly), and confirmed it's not an obligatory intermediate in alpha- to gamma-thrombin conversion.
- Observed simultaneous minor cleavages and demonstrated comparable proteolytic activity between alpha-thrombin and a mixture of autolysed forms (beta-, beta'-, and gamma-thrombins).
Conclusions:
- Autolysis of human alpha-thrombin is a complex process involving multiple sites and intermediates.
- Beta'-thrombin represents a new intermediate in thrombin autolysis.
- Autolysed thrombin retains significant proteolytic activity and specificity, comparable to the native enzyme.
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