Question

Data Analysis Problem by Marianna Pap and József Szeberényi to accompany The Cell: A Molecular Approach, Seventh Edition Geoffrey M. Cooper and Robert E. Hausman 2.3 The Effect of a Reducing Agent on Protein Structure Source: Janatova, J. 1986. Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement. Biochem, J. 233: 819-825. Level of difficulty: Medium Corresponding chapter(s) in the textbook: Chapter 2 Review the following terms before working on the problem: tertiary and quaternary protein structure, chemical bonds, reducing agents, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), protein staining EXPERIMENT This experiment was designed to analyze the structure and electrophoretic behavior of a component (called C3c protein) of the complement system. The complement system consists of various proteins in the blood plasma of vertebrates and is involved in the immune response against microorganisms. Purified C3c protein (molecular mass: 145 kD) was incubated in the presence of various concentrations of dithiothreitol (DTT), a reducing agent, and then subjected to SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Brillant Blue, a protein dye. The figure shows the molecula masses of intermediates and products generated by DTT treatment.

Answer 1

1. 3 polypeptides are present having sizes 75, 43 and 27 kDa.

2. DTT is used as a reducing agent since it functions in reducing the disulfide bonds formed in the proteins and keeps the intermolecular and intramolecular cysteines in reduced form prevent them from getting oxidised to form disulfide linkages.

Complement proteins are highly cysteine rich in nature and due to high cysteine content the chances of formation of inter and intramolecular disulfide linkages are more due to oxidation of those free cysteine groups.

3. Various kinds of bonds such as Hybrophobic interactions, disulfide bonds, Hydrogen bonds , ionic interactions areinvolved in keeping proteins/polypeptides together.

In this cases the majority of forces are disulfide linkages both inter and intradisulfide linkages hold the complement proteins together.

4. 102 kD band is the result of formation of disulfide linkages between free cysteines by their oxidation. As the concentration of DTT is further increased at 10mM , Then this band disappears and the free cysteines are kept in highly reduced state not allowing then to form this oxidation product. This was a polymeric disulfide linked product formed due to cysteine oxidation.