14. Assume you have isolated a relatively abundant protein, and you want to obta

Question

14. Assume you have isolated a relatively abundant protein, and you want to obtain the amino acid sequence. You added dithiothreitol to the sample then did SDS-PAGE and found two bands A at 2.9 and B at 3.9 kD,. You then cleaved the protein with chymotrypsin and found the following fragments: Band A CN Band B NLQNY GIVEQCCHKRCSEY DPTKM ACGVRGF RTTGHLCGKDLVNALY You then cleaved with Staphylococcus aureus V8 protease and found the following PTKM RTTGHLCGKD LVNALYIACGVRGFFYD Band B Band A GIVE YNLQNYCN QCCHKRCSE What is the primary amino acid sequence of the protein?

Explanation / Answer

Chymotrypsin cleaves only at the carboxyl ends of phe, trp, tyr and leu residues provided that proline is not the nect amino acid in the sequence. Whereas, S. aureus protease cleaves only at the carboxyl end of asp and glu residues.

Considering the abovementioned facts in each cases for both bands,

the sequence of A will be GIVEQCCHKRCSEYNLQNYCN

the sequence of B will be RTTGHLCGKDLVNALYIACGVRGFFYDPTKM

These two bands will be connected with each other through a disulfide bridge with the aid of cys residue present in each sequence. The disulfide bond was reduced to -SH groups after treatment with DTT. That's why two bands appeared.

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