Abstract
Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.
Highlights
Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions
This reaction may beinvolved in the normal aging of tissues because the extent of protein glycation increases with age in man [20], possibly in response to decreased protein turnoverand thedevelopment of glucose intolerance
Knowledge of the factors affecting the specificity of protein glycation may be important in understanding the role of this chemical modification in the structuraalnd functional changes in protein which occur both in diabetes and during normal aging
Summary
Kinetics of Modification of RNase by Glucose-RNase was incubated with glucose in order to study both itsrate of reaction with glucose and the effect of glycation on its enzymatic activity. When the glycation reaction is carried out in the presence of NaBH3CN, this reagent traps theSchiff base intermediate by reducing it directly to glucitollysine (Fig. 1).Proteins are modified more rapidly in this case because the rate of formation and reduction of the Schiff base is more rapid than that of the Amadori rearrangement [1,3]. During the early stages of the reaction in the presence of NaBH3CN, before extensive modification of the protein had occurred, the rates of modification of lysine residues and loss of enzymatic activity were similar. Initial Sites of Formation of Amadori Adducts to RNaseThe sites of modification of RNase by glucose wereexamined by tryptic peptide analysis of RNase modified with 1mol of Glc/mol of protein, i.e. after 3 days incubation with glucose '\.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
