Thiols enhance the sensitivity of luminescent assays for non-cross-linked and covalently cross-linked aminophthalhydrazides.
Transglutaminases catalyse an acyl-transfer reaction between the gamma-carboxamide of a protein or peptide-bound glutamine (P-CH2-gammaCH2-CO-1NH2) and the primary amino group of mono- or polyamines (R-2NH2), covalently cross-linking the reactants by an isopeptide bond: P-CH2-gammaCH2-CO-1NH2 + R-2NH2 --> P-CH2-gammaCH2-CO-2NH-R + 1NH3. We reported that N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was a chemiluminescent (CL) amine substrate for transglutaminases. We now identified N-(6-aminohexyl)-N-ethylisoluminol (AHEI) as a second, less reactive, transglutaminase substrate. A structure-based explanation is offered for the lower reactivity of AHEI. Optimum CL from non-cross-linked or cross-linked ABEI or AHEI was elicited in the presence of 10 mmol/L dithiothreitol, by oxidizing with a mixture of 20 mmol/L potassium ferricyanide and 10 mmol/L hydrogen peroxide in 100 mmol/L NaOH. The limits of detection and quantitation for non-cross-linked aminophthalhydrazides obtained in this system were: 20 fmol and 60 fmol for ABEI and 10 fmol and 30 fmol for AHEI. These values represented a 500-800-fold improved sensitivity. Delayed peak CL and CL decay in the presence of dithiothreitol contributed to improving the sensitivity. The data could be useful for improving the immunoassays for aminophthalhydrazides and facilitate the development of high throughput assays for transglutaminases.
Achyuthan
K E
KE
ZymeTx, Inc., 800 Research Parkway 100, Oklahoma City, OK 73104-3600, USA. achyuthank@zymetx.com
D000818:Animals; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008163:Luminescent Measurements; D008165:Luminol / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010793:Phthalazines / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D011503:Transglutaminases / Q000378:metabolism
D003432:Cross-Linking Reagents; C446501:N-(6-aminohexyl)-N-ethylisoluminol; D010793:Phthalazines; D013438:Sulfhydryl Compounds; C032487:isoluminol; D008165:Luminol; D011503:Transglutaminases