Question

1) a. Write two methods that can be used to determine protein primary structure b. what is the purpose of CNBr and betamercaptoethanol in determining protein primary structure?

Answer 1

Amino acid sequence of the proteins. The convention for the designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the free amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i.e. the end with the residue containing a free -carboxyl group) is to the right. Here we use several methods to determine the structure of protein with the help of N terminal and C-terminal of amino acids

1)Sanger' s method

2)Edman's method

PURPOSES OF

A) CYANOGEN BROMIDE - Cyanogen bromide (CNBr) cleaves at methionine (Met) residues through its Carbon side.

For example in sequence like, Asp-Arg-met-val when we hydrolyzed it with cyanogen bromide it gives 1)Asp-Arg-Met 2)Val

B) betamarcaptoethanol-

Some proteins can be denatured by 2-mercaptoethanol, which cleaves the disulfide bonds that may form between thiol groups of cysteine residues. In the case of excess 2-mercaptoethanol, the following equilibrium is shifted to the right:

By breaking the S-S bonds, both the tertiary structure and the quaternary structure of some proteins can be disrupted.[6] Because of its ability to disrupt the structure of proteins, it was used in the analysis of proteins, for instance, to ensure that a protein solution contains monomeric protein molecules, instead of disulfide linked dimers or higher order oligomers.