title: Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.
abstract: Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37 °C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A.
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Dinda
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India.
authors.forename: Debi Ranjan
authors.initials: DR
authors.lastname: Tripathy
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India. Electronic address: swagata@chem.iitkgp.ernet.in.
authors.forename: Swagata
authors.initials: S
authors.lastname: Dasgupta
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017127:Glycation End Products, Advanced; D005632:Fructose; D012266:Ribose; D004247:DNA; D012259:Ribonuclease, Pancreatic; D005947:Glucose
keywords: AGE cross-linking; Advanced glycation end products (AGEs); DNA melting; Ribonuclease A; Ribonuclease inhibitor; Ribonucleolytic activity