Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o-phthaldialdehyde reagent.

Abstract

The potential application of the o-phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH3, a by-product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH3 (at NH3 concentrations > 10 mM) was found to decrease during extended incubation at 23, 37 and 50 degrees C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH3 from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mm NH3 at 23 degrees C and pH 7.0 was found to be 88%. The release of NH3 and the decrease in epsilon-amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23 degrees C resulted in progressive increases and decreases, respectively, in NH3 and -amino group concentration with increased incubation time. These changes were dependent on TGase:NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase-catalysed cross-links. However, the observed decrease in epsilon-amino group concentration was higher than expected. This may be due to concealment of noncross-linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.