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Biochim Biophys Acta. 1980 Aug 7;614(2):339-42.

A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase.


We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.

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