Date of Award

2015

Degree Name

Chemistry

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Leslie Frost

Second Advisor

Derrick Kolling

Third Advisor

Bin Wang

Abstract

For this research project, I propose a method for the analysis of glycated lysozyme using Matrix Associated Laser Desorption / Ionization-Time of Flight Mass spectrometry (MALDI-TOF MS). Glycation or "Maillard reaction" is a non-enzymatic reaction that occurs between amino acid residues, usually primary and secondary amines, of proteins and the carbonyl group of reducing sugars to yield Schiff bases. The glycation mechanism occurs between the carbonyl group of the reducing sugars and a primary amine by nucleophilic addition, resulting in the formation of a Schiff base. The Schiff base can then rearrange into the more stable Amadori product which is the first level of protein glycation. By processes such as oxidation, dehydration, and condensation, the Amadori product rearranges in time to form a heterogeneous group of compounds referred to advanced glycation end products (AGEs). Carnosine, a dipeptide consisting of β-alanine and histidine, has been shown to have anti-glycating characteristics that can hinder the damaging impact of aldo- and keto-sugars through oxidation, and it also defends against such age-stimulating processes as glycation, oxidation, and protein cross-linking. Lysozyme is an enzyme capable of destroying the bacterial cell wall, and it acts as protection from bacterial infection. The goal of this project was to determine the initial glycation sites on chicken and human lysozyme in vitro, determine how carnosine affects the initial glycation of lysozyme and the formation of AGE structures, and investigate how glycation affects the enzymatic activity of the enzyme. Lysozyme was incubated with D-glucose and D-galactose for three days at 37°C in the presence and absence of carnosine and analyzed by MALDI-TOF mass spectrometry. Lysozyme was primarily found to condense with one sugar molecule, and the preferred amino acid site of glycation for chicken lysozyme was identified as Lysine-115 which is not located near the catalytic cleft of the enzyme. In fact, the enzymatic activity of chicken lysozyme did not change significantly when it was glycated using my glycation conditions. When both chicken and human lysozyme was incubated with sugars in the presence of carnosine, I found that carnosine did not inhibit the initial glycation reaction between lysozyme and Dgalactose or D-glucose. The final experiment was to determine the effect of carnosine on the formation of AGE structures on both chicken and human lysozyme after the initial glycation reaction had occurred. The results of these experiments indicated that carnosine can decelerate or hinder the formation of AGE structures that can be obtained after the initial glycation of the protein.

Subject(s)

Lysozyme - Research.

Glycosylation - Research.

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