Simultaneous activity assay of two transglutaminase isozymes, blood coagulation factor XIII and transglutaminase 2, by use of fibrinogen arrays.

Abstract

We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.

• Authors: Mi-Hye Kwon, Se-Hui Jung, Young-Myeong Kim, Kwon-Soo Ha
• Journal: Analytical chemistry (Anal Chem) Vol. 83 Issue 22 Pg. 8718-24 (Nov 15 2011) ISSN: 1520-6882 [Electronic] United States
• PMID: 21961727 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Isoenzymes
• Fibrinogen
• Factor XIII
• Protein Glutamine gamma Glutamyltransferase 2
• Transglutaminases
• GTP-Binding Proteins

Topics

• Animals
• Factor XIII (metabolism)
• Fibrinogen
• GTP-Binding Proteins (metabolism)
• Guinea Pigs
• High-Throughput Screening Assays
• Humans
• Isoenzymes (metabolism)
• Protein Glutamine gamma Glutamyltransferase 2
• Transglutaminases (metabolism)
• Tumor Cells, Cultured