Date of Award

1-1-1983

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

The 15 surface carboxyl groups in alpha-chymotrypsin first were modified with glycine ethyl ester (Gly-OEt) and taurine (Tau) at pH 4 using carbodiimide (EDC) reagent to give G(,15)-Chy and T(,15)-Chy, respectively. These enzymes (Chy-15) were remodified using norleucine methyl ester (Nle-OMe) as a tag in the presence of 8M urea. The analytical determination of the Nle-OMe tag was done by amino acid analysis and showed 1.5 and 2 moles of Nle per mole of T(,15)-Chy and G(,15)-Chy, respectively. Initial modification of alpha-chymotrypsin in the presence of 3M urea using Tau and Gly-OEt show 15.8 and 16 moles of Tau and Gly per mole of enzyme, respectively. Subjecting T(,16)-Chy and G(,16)-Chy to further modification in the presence of 8M urea using Nle-OMe showed 1 mole of Nle per mole. When native alpha-chymotrypsin was modified in the presence of 8M urea 17 moles of these adducts were found per mole.The specificities of selectively modified enzymes were assayed using dipeptides and alpha-amino acid esters. The enzymes with only the surface carboxyls blocked (Chy-15) behaved similar to native chymotrypsin. When in addition to the surface carboxyls, one buried carboxyl was modified (Chy-16), the enzymes were found to be unreactive with dipeptide substrates, but reactive towards simple amino acid esters.Conventional titration methods were used to find the pKa values of the unmodified carboxyls in modified enzymes. In the presence of 0.1M NaCl and SDS (Lauryl), the unmodified carboxyls in Chy-15 derivatives had pKa values of about 5 and 6, and Chy-16 derivatives had one ionizable group with pKa near 5.In the absence of SDS, Fourier transform infrared spectroscopy (FT-IR) was used to determine the pKa values of the unmodified carboxyls. Without salt G(,15)-Chy and T(,15)-Chy showed two carboxyls with pKa's of 6 and 7, whereas G(,16)-Chy and T(,16)-Chy had only one pKa of 7.2-7.3. In the presence of salt, the same pKa values were shifted lower to 5.8, 6.8, and 6.2, respectively.The use of norleucine and p-nitroanilide tags indicate that the first buried carboxyl to be modified is located in the B-chain (Asp-102).

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