Flow-injection spectrophotometric determination of acetyl-coenzyme A with immobilized phosphotransacetylase |
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Authors: | Susumu Yamato Kenji Shimada |
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Institution: | Department of Analytical Chemistry, Niigata College of Pharmacy, Niigata 950-21 Japan |
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Abstract: | 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. |
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