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Updated: May 24, 2026
Detection of Functional Matrix Metalloproteinases by Zymography
Published on: November 8, 2010
Matrix metalloproteinase-9 activity detected in body fluids is the result of two different enzyme forms
Tiziana Bellini1, Alessandro Trentini, Maria C Manfrinato
1Section of Biochemistry and Clinical Biochemistry, Department of Biochemistry and Molecular Biology, University of Ferrara, Via Luigi Borsari 46, Italy.
Abstract:
In vitro activation of matrix metalloproteinase-9 (MMP-9) (Gelatinase B) with MMP-3 shows the presence of two different forms: an 82 kDa, N-terminal truncated form, and a 65 kDa, N- and C-terminal truncated form. So far the presence of the 65 kDa form has not been reported in vivo. Affinity chromatography was performed to separate MMP-9 from MMP-2 and immunoprecipitation to isolate ∼65 kDa MMP-9 from 82 kDa MMP-9 in sera of healthy donors. The presence of ∼65 kDa active MMP-9 was demonstrated both with gelatin zymography and western blot analysis. The ∼65 kDa MMP-9 lacks the haemopexin domain required for the high-affinity binding of the tissue inhibitor TIMP-1, and can be evaluated by activity assay in the presence of TIMP-1. This opens the possibility to investigate the role of this form of MMP-9 that escapes physiological regulation.
Insights
Researchers identified a novel, active 65 kDa form of matrix metalloproteinase-9 (MMP-9) in human blood. This MMP-9 form lacks a key regulatory domain, suggesting it may evade normal physiological control.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Matrix metalloproteinase-9 (MMP-9), also known as Gelatinase B, is an enzyme involved in extracellular matrix degradation.
- In vitro studies have identified different activated forms of MMP-9, including 82 kDa and 65 kDa truncated variants.
- The in vivo presence and significance of the 65 kDa MMP-9 form have not been previously established.
Purpose of the Study:
- To investigate the in vivo presence of the truncated 65 kDa active form of MMP-9 in human sera.
- To characterize the biochemical properties of the 65 kDa MMP-9 form.
- To explore the implications of a potentially unregulated MMP-9 form in physiological processes.
Main Methods:
- Affinity chromatography was employed to separate MMP-9 from MMP-2.
- Immunoprecipitation was used to isolate the approximately 65 kDa MMP-9 from the 82 kDa form in sera of healthy donors.
- Gelatin zymography and western blot analysis were utilized to confirm the presence and activity of the 65 kDa MMP-9.
Main Results:
- The study successfully isolated and identified the approximately 65 kDa active MMP-9 form in sera from healthy individuals.
- This 65 kDa MMP-9 variant was found to lack the haemopexin domain, which is crucial for binding to TIMP-1.
- The absence of the haemopexin domain allows for activity assays in the presence of TIMP-1, indicating it bypasses this inhibitory regulation.
Conclusions:
- A novel, biologically active 65 kDa form of MMP-9 exists in vivo in healthy individuals.
- This MMP-9 variant's lack of the haemopexin domain suggests it may escape physiological regulation by TIMP-1.
- Further research is warranted to elucidate the specific roles and implications of this unique MMP-9 form in health and disease.
