Analytical Approaches to Alcohol Dehydrogenase Structures
Sequence analysis of polypeptides by Edman degradation is dependent on a free N-terminal α-amino group for the reaction with phenylisothiocyanate. However, alcohol dehydrogenases, like many other proteins, contain an acetylated N-terminal residue which blocks degradation (cf. Tsunasawa and Hirano, 1993). The conventional approach for blocked proteins involves enzymatic or chemical cleavage and reverse-phase HPLC-sepa-ration of fragments, followed by internal sequence analysis. The drawbacks associated with this technique are high protein consumption, long handling times and the fact that the N-terminal fragment remains inaccessible to Edman degradation. In this paper, we have tested direct chemical deblocking and applied it to both a synthetic peptide corresponding to the N-terminal segment of horse liver alcohol dehydrogenase and to the intact protein.
KeywordsAlcohol Dehydrogenase Intact Protein Magnetic Circular Dichroism Edman Degradation Electro Spray Ionization
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