Question

After the polypeptide shown below was treated with maleic anhydride, it was hydrolyzed by trypsin. (After a polypeptide is treated with maleic anhydride,

trypsin will cleave the polypeptide only on the C-side of arginine.)

Gly-Ala-Asp-Ala-Leu-Pro-Gly-Ile-Leu-Val-Arg-Asp-Val-Gly-Lys-Val-Glu-Val-Phe-Glu-Ala-Gly-

Arg-Ala-Glu-Phe-Lys-Glu-Pro-Arg-Leu-Val-Met-Lys-Val-Glu-Gly-Arg-Pro-Val-Gly-Ala-Gly-Leu-Trp

a. After a polypeptide is treated with maleic anhydride, why does trypsin no longer cleave it on the C-side of lysine?

b. How many fragments are obtained from the polypeptide?

c. In what order will the fragments be eluted from an anion-exchange column using a buffer of pH = 5?

Short answer

a. Trypsin will cleave only at arginine residues as the enzyme will no longer recognize lysine residues.

b. Four fragments are obtained from the polypeptide.

c.The elution order is as follows:

A>D>C>B.

A Gly-Ala-Asp-Ala-Leu-Pro-Gly-Ile-Leu-Val-Arg overall charge = 0

+ - + -

B Asp-Val-Gly-Lys-Val-Glu-Val-Phe-Glu-Ala-Gly-Arg overall charge = -3

+ - - - - +-

C Ala-Glu-Phe-Lys-Glu-Pro-Arg overall charge = -2

+ - - - + -

D Leu-Val-Met-Lys-Val-Glu-Gly-Arg-Pro-Val-Gly-Ala-Gly-Leu-Trp overall charge=-1

+ - - + -

Step-by-step solution

Polypeptide

Polypeptide constitutes biomaterials comprising repeating amino acid units connected by a peptide bond. One or several polypeptide chains form protein. Each polypeptide chain comprises several amino acids connected.

The treatment of polypeptide with maleic anhydride

a. When the polypeptide is reacted with maleic anhydride, lysine reacts with maleic anhydride, but the amino group of arginine is not nucleophilic to react with maleic anhydride.

Hence, trypsin will cleave only at arginine residues as the enzyme will no longer recognize lysine residues.

b. Four fragments are obtained from the polypeptide. Trypsin will not cleave the Arg-Pro bond.

c. The N-terminal end of each fragment is positively charged because of the group. The C-terminal end is negatively charged because of the COO^- group. The arginine residues are positively charged.

Aspartate and glutamate residues are negatively charged. Lysine residues are negatively charged because they are connected to the maleic acid group.

The elution order is as follows:

A>D>C>B.

A Gly-Ala-Asp-Ala-Leu-Pro-Gly-Ile-Leu-Val-Arg overall charge=0

+ - + -

B Asp-Val-Gly-Lys-Val-Glu-Val-Phe-Glu-Ala-Gly-Arg overall charge=-3

+ - - - - +-

C Ala-Glu-Phe-Lys-Glu-Pro-Arg overall charge= -2

+ - - - + -

D Leu-Val-Met-Lys-Val-Glu-Gly-Arg-Pro-Val-Gly-Ala-Gly-Leu-Trp overall charge=-1

+ - - + -