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

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°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°C and pH 7.0 was found to be 88%. The release of NH₃ and the decrease in ε-amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23°C resulted in progressive increases and decreases, respectively, in NH₃ and ε-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 ε-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.