Abstract
It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation.
Highlights
Perhaps the most stable proteins in the body are ␣, , and ␥ crystallins found in the lens
As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as ␥-S crystallin that were synthesized during the fetal-embryonic periods of development
Because the  and ␥ crystallins belong to a different family of lens proteins, and because they comprise the majority of proteins present in the dry weight material of the lens, it is important to verify whether asparagine and glutamine residues in this class of lens proteins are relatively resistant to in vivo deamidation
Summary
Normal human lenses ages 57, 60, and 66 years were obtained from the National Disease Research Interchange, Philadelphia, PA. After thawing the lens at room temperature, the fetal-embryonic region of each lens was obtained by dissection (3). This region of the lens was dissolved completely in 7 M guanidine hydrochloride, 10 mM EDTA, and 0.3 M Tris-hydrochloride, pH 8.6, and was reduced by carboxymethylation and dialyzed against distilled water as described previously (2). Protein was determined as described previously (4) using bovine serum albumin as standard.
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