A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate.
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.
Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710.
T F
TF
Slaughter
K E
KE
Achyuthan
T S
TS
Lai
C S
CS
Greenberg
D000588:Amines; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005176:Factor XIII / Q000032:analysis / Q000235:genetics; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology; D015335:Molecular Probes; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D011503:Transglutaminases / Q000032:analysis* / Q000037:antagonists & inhibitors
D000588:Amines; D015335:Molecular Probes; D011994:Recombinant Proteins; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D005176:Factor XIII; D011503:Transglutaminases