Microheterogeneity of human antithrombin III

Area of Science:

• Biochemistry
• Proteomics
• Glycobiology

Background:

• Antithrombin is a crucial plasma protein regulating coagulation.
• Understanding antithrombin heterogeneity is vital for its therapeutic applications.
• Previous studies indicated potential variations in antithrombin structure.

Purpose of the Study:

• To investigate the microheterogeneity of purified human plasma antithrombin.
• To identify the molecular basis for observed differences in antithrombin fractions.
• To assess the impact of glycosylation on antithrombin properties.

Main Methods:

• Isoelectric focusing in a pH 4-6 gradient to separate antithrombin fractions.
• Polyacrylamide gel electrophoresis (PAGE) to assess homogeneity.
• Amino acid composition analysis.
• Specific activity assays.
• Cross-reactivity tests with antithrombin antiserum.
• Analysis of sialic acid and aminosugar content.
• Neuraminidase treatment followed by isoelectric focusing.

Main Results:

• Two homogeneous antithrombin fractions were isolated by isoelectric focusing.
• Both fractions exhibited similar amino acid composition, specific activity, and immunoreactivity.
• Significant differences in sialic acid and aminosugar content were detected between the fractions.
• Neuraminidase treatment resulted in a single homogeneous peak with preserved antithrombin activity.

Conclusions:

• The microheterogeneity of human plasma antithrombin is primarily attributed to variations in glycosylation.
• Differences in sialic acid content contribute to the observed charge heterogeneity.
• Antithrombin activity remains stable despite alterations in glycosylation patterns.