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Non-Enzymatic Protein Acetylation by 7-acetoxy-4-methylcoumarin: Implications in Protein Biochemistry

Author(s):

Anant Narayan Bhatt *, Yogesh Rai, Amit Verma, Sanjay Pandey, Kumar Kaushik, Virinder S Parmar, Anu Arya, Ashok K Prasad and Bilikere S Dwarakanath   Pages 1 - 8 ( 8 )

Abstract:


Background: The semi-synthetic acetoxycoumarins are known to acetylate proteins using novel enzymatic calreticulin transacetylase (CRTAase) system in cells. However, the non-enzymatic protein acetylation by polyphenolic acetates is not known.

Objective: To investigate the ability of 7-acetoxy-4-methyl coumarin (7-AMC) to acetylate proteins non-enzymatically in the test tube.

Methods: We incubated 7-AMC with BSA and analyzed the protein acetylation using western blot technique. Further, BSA induced biophysical changes in the spectroscopic properties of 7-AMC was analyzed using Fluorescence spectroscopy.

Results: Using pan anti-acetyl lysine anti┬Čbody, herein we demonstrate that 7-AMC acetylates bovine serum albumin (BSA) in time and concentration dependent manner in the absence of any enzyme. 7-AMC is a relatively less fluorescent molecule compared to the parental compound, 7-hydroxy-4-methylcoumarin (7-HMC), however the fluorescence of 7-AMC increased by two fold on incubation with BSA, depending on the time of incubation and concentration of BSA. Analysis of the reaction mixture of 7-AMC and BSA after filtration revealed that the increased fluorescence is associated with the compound of lower molecular weight in the filtrate and not residual BSA, suggesting that the less fluorescent 7-AMC undergoes self-hydrolysis in the presence of protein to give highly fluorescent parental molecule 7-HMC and acetate ion in polar solvent (phosphate buffered saline, PBS). The protein augmented conversion of 7-AMC to 7-HMC was found to be linearly related to the protein concentration.

Conclusion: Thus protein acetylation induced by 7-AMC could also be non-enzymatic in nature and this molecule can be exploited for quantification of proteins.

Keywords:

Protein acetylation, 7-AMC, polyphenolic acetates, coumarins , BSA , Fluorescence

Affiliation:

Institute of Nuclear Medicine and Allied Sciences, Metabolic and Cell Signaling Research, Brig. SK Mazumdar Mrg, Timarpur, Institute of Nuclear Medicine and Allied Sciences, Metabolic and Cell Signaling Research, Brig. SK Mazumdar Marg, Timarpur, Institute of Nuclear Medicine and Allied Sciences, Metabolic and Cell Signaling Research, Institute of Nuclear Medicine and Allied Sciences, Brig. SK Mazumdar Mrg, Timarpur, Institute of Nuclear Medicine and Allied Sciences, Metabolic and Cell Signaling Research, Medgar Evers College, The City University of New York, Department of Chemistry and Environmental Science, Medgar Evers College, The City University of New York, Department of Chemistry and Environmental Science, V.P.Chest Institute, Dept of Biochemistry, University of Delhi, Dept of Chemistry, Institute of Nuclear Medicine and Allied Sciences, Metabolic and Cell Signaling Research



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