Thiols enhance the sensitivity of luminescent assays for non-cross-linked and covalently cross-linked aminophthalhydrazides.

K E Achyuthan

Index: Luminescence 16(4) , 257-62, (2001)

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Abstract

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.Copyright 2001 John Wiley & Sons, Ltd.