Analysis of carbamylation and acetylation of lens proteins

Wen Qin, Purdue University

Abstract

The reactivities of some amino acid residues in crystallins which undergo modifications with cyanate and aspirin were investigated in this study. Bovine lens $\alpha$A- and $\gamma$II-crystallins were used for this study because both crystallins have been found in cataractous lens homogenate as high molecular weight aggregates. $\alpha$-crystallins were incubated with cyanate and separated into $\alpha$A and $\alpha$B-crystallins by reversed phase HPLC. Products of the reaction were analyzed by HPLC and FABMS to determine the extent of reaction of each of the seven lysyl residues present in $\alpha$A. Results demonstrated that incubation of $\alpha$-crystallins with cyanate leads to partial carbamylation of all 7 lysines in $\alpha$A. Rate constants for the reaction of specific lysyl residues with cyanate ranged from 0.05 to 0.54 M$\sp{-1}$h$\sp{-1}$. The distribution of reaction products, as determined by isoelectric focusing, indicated that the physiologically relevant initial stages of carbamylation of the seven lysyl residues of $\alpha$A proceed in a noncooperative manner. $\gamma$II-crystallin was digested with trypsin in the presence of 20% CH$\sb3$CN, which allowed us to map the entire sequence. The experimental data showed a Thr rather than a Ser at position 119. $\gamma$II-crystallin was incubated with cyanate and aspirin at pH 7.4 and the products of the reactions were analyzed by FABMS. Rate constants for carbamylation and acetylation of the N-terminus and both Lys 2 and Lys 163 were determined. Results indicated that aspirin reacted with the amino groups in a similar fashion as cyanate. Our investigations of the reactions of cyanate and aspirin with $\gamma$II-crystallin showed that the cysteinyl residues are also carbamylated and acetylated. Stability studies of the S-modified peptides at pH 7.4 indicated that, at this pH, the carbamylation of the cysteinyl residues was reversible, leading to regeneration of the thiol group and disulfide bonding, and that the acetylated cystein residues was stable and prevented disulfide bonding. This difference in stability explains how cyanate promotes, and aspirin inhibits, cataractogenesis.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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