| Autor: |
Fowler, Elizabeth, Moyer, Mary, Krishna, Radha G., Chin, Christopher C.Q., Wold, Finn |
| Zdroj: |
Current Protocols in Protein Science; March 1996, Vol. 3 Issue: 1 p11.7.1-11.7.17 |
| Abstrakt: |
Two enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A [Colorimetric Assay for Pyroglutamate Aminopeptidase Activity] describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N‐terminally blocked proteins using acid‐catalyzed hydrolysis or methanolysis, hydrazinolysis, and β‐elimination after acid‐catalyzed N‐O shift. Alternate protocols describe chemical removal of acetyl and longer‐chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate. |
| Databáze: |
Supplemental Index |
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