Question

xplain the purpose/function of each of the following reagents used in the isolation of the iron containing protein: (4 points each)

Chymotrypsin

Hydroxylamine hydrochloride

1,10‐phenanthroline

Ethanol

Trichloroacetic acid

Answer 1

Function of Chymotrypsin - Chymotrypsin is a digestive enzyme component of pancreatic juice acting in the duodenum where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P₁ position) is a large hydrophobic amino acid (tyrosine,tryptophan, and phenylalanine).

Function of Hydroxylamine hydrochloride - Hydroxylamine hydrochloride can be used to highly selectively cleave asparaginyl - glycine peptide bonds in peptides and proteins.

Function of 1,10‐phenanthroline - 1,10-Phenanthroline is an inhibitor of metallopeptidases, with one of the first observed instances reported in carboxypeptidase A. Inhibition of the enzyme occurs by removal and chelation of the metal ion required for catalytic activity, leaving an inactive apoenzyme. 1,10-Phenanthroline targets mainly zinc metallopeptidases, with a much lower affinity for calcium.

Function of Trichloroacetic acid - The trichloroacetic acid (TCA) - insoluble fraction of water column bacteria labeled with leucine contained an ethanol - soluble fraction accounting for up to 44% of the label. A component of the ethanol - soluble fraction is leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins.