Flow-injection spectrophotometric determination of acetyl-coenzyme A with immobilized phosphotransacetylase

Phosphotransacetylase (PTA) is immobilized on AF-Tresyl TOYOPEARL 650 gel and used on-line in a stainless-steel column (10 × 4 mm i.d.). CoA-SH liberated enzymatically from acetyl-CoA is reacted with Ellman's reagent 5,5′-dithiobis(2-nitrobenzoic acid)] in the carrier stream. The calibration graph for acetyl-CoA is linear from 4 × 10^?6 to 4 × 10^?4 M and the detection limits is 8 × 10^?7 M. The immobilized enzyme can be employed for over 4 months without any significant decrease in activity. The enzyme retains its activity in methanol even though the initial rate of reaction is decreased.