A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate

Anal Biochem. 1992 Aug 15;205(1):166-71. doi: 10.1016/0003-2697(92)90594-w.


Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein. The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product is detected by streptavidin-alkaline phosphatase and quantitated by measuring the absorbance at 405 nm following the addition of p-nitrophenyl phosphate. The assay is sensitive, specific, and linear at plasma factor XIIIa concentrations between 0.08 and 1.25 micrograms/ml and at purified guinea pig liver transglutaminase concentrations between 0.05 and 0.8 microgram/ml. The intra-assay coefficient of variation is less than 8%. The solid-phase assay was used to quantitate the transglutaminase activity in Escherichia coli extracts expressing recombinant factor XIII A-chains and to analyze factor XIIIa inhibitors. This method will facilitate the analysis of structure-function relationships of the transglutaminases using recombinant DNA methods. Furthermore, screening of natural and synthetic factor XIIIa inhibitors will be expedited by this solid-phase microtiter plate assay.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amines*
  • Animals
  • Biotin / analogs & derivatives*
  • Chemistry Techniques, Analytical / methods*
  • Escherichia coli / genetics
  • Factor XIII / analysis
  • Factor XIII / genetics
  • Guinea Pigs
  • Liver / enzymology
  • Molecular Probes
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Structure-Activity Relationship
  • Substrate Specificity
  • Transglutaminases / analysis*
  • Transglutaminases / antagonists & inhibitors


  • Amines
  • Molecular Probes
  • Recombinant Proteins
  • 5-(biotinamido)pentylamine
  • Biotin
  • Factor XIII
  • Transglutaminases