lecture 24氨基酸,多肽和蛋白质.pptVIP

* * * * * * * * * * * * * * * * * * * * * * * * * * Copyright ? 2006 Pearson Prentice Hall, Inc. * * * * * * * * * * * * * * * * * * Composition of Human Bradykinin Use of an amino acid analyzer to determine the composition of human bradykinin（舒缓激肽）. Sequencing from the N Terminus: The Edman Degradation Edman degradation: The reaction with phenyl isothiocyanate followed by hydrolysis removes the N terminus amino acid. The phenylthiohydantoin derivative is identified by chromatography. Use for peptides with 30 amino acids. Edman Degradation Last Step of the Edman Degradation In the final step (step 3) the thiazoline isomerizes to the more stable phenylthiohydantoin. 色谱检测到的噻唑环就是N-端氨基酸 The Sanger Method The Sanger method for N terminus determination is a less common alternative to the Edman degradation. 系统中含有2,4-二硝基苯基的氨基酸就是氮端氨基酸。 Sequencing from the C Terminus The enzyme carboxypeptidase（羧肽酶） cleaves the C-terminal peptide bond. However, since different amino acids react at different rates, it’s difficult to determine more than the original C- terminal amino acid. C-Terminal Residue Analysis The C-terminal amino acid can be identified using the enzyme carboxypeptidase, which cleaves the C-terminal peptide bond. Eventually, the entire peptide is hydrolyzed to its individual amino acids. Partial Hydrolysis Break the peptide chain into smaller fragments. Trypsin（胰蛋白酶） cleaves at the carboxyl group of lysine and arginine. Chymotrypsin（胰凝乳蛋白酶） cleaves at the carboxyl group of phenylalanine, tyrosine（酪氨酸）, and tryptophan（色氨酸）. Sequence each fragment, then fit them together like a jigsaw puzzle（七巧板拼图）. Solution Phase Peptide Synthesis: Preliminary Step Protect the amino group with Z. Solution Phase Peptide Synthesis: Step 1 Activate the carboxyl group with ethyl chloroformate to form anhydride of carbonic acid. Solution Phase Peptide Synthesis: Step 2 Form an amide bond to couple the next amino acid Solution Phase Peptide Synthesis: Final Step Remove the protec