Title:Non-Enzymatic Protein Acetylation by 7-Acetoxy-4-Methylcoumarin: Implications in Protein Biochemistry
Volume: 27
Issue: 8
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*
Affiliation:
- Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Majumdar Marg, Timarpur, Delhi,India
- Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Majumdar Marg, Timarpur, Delhi,India
Keywords:
Protein acetylation, 7-AMC, polyphenolic acetates, coumarins, BSA, fluorescence.
Abstract:
Background: The semi-synthetic acetoxycoumarins are known to acetylate proteins
using novel enzymatic Calreticulin Transacetylase (CRTAase) system in cells. However, the nonenzymatic
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 antibody, 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.