Review the following terms before working on the problem: tertiary and quaternar

Question

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 molecular masses of intermediates and products generated by DTT treatment. FIGURE 102 QUESTIONS 1. How many polypeptides are present in C3c? Determine their molecular masses from the figure.

Explanation / Answer

Answer-

1) Four different types of bands are there in gel (in figure) and the molecular weights are 102, 75, 42 and 27 Kd.

2) The purpose of usig DTT in this experiment is to prevent oxydation of thiol groups as DTT is used as protecting reagents. DTT further denatues proteins by reducing there disulphide bonds.

3) Polypeptide bonds hold the polypeptide together

4) 102 Kd polypeptide band is larger than the other band sizes ( 72, 43 and 27)

5) The 43 Kd band showed up as two bands. The precursor-product means, one of them is precursor which is little higher molecular weight band which get cleaved to become product. hence two bands appear in SDS-PAGE at 43 kd.

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