Looking for help with questions B and C Thanks The amino aci

The amino acid sequence of BN was finally determined in the following manner: The BN protein was first treated with beta-mercaptoethanol. This treatment revealed that the BN protein consisted of two chains, a light chain and a heavy chain^2. Next, the light chain and heavy chain were separated and then three separate samples of the purified chains were treated with three different proteases. The fragments obtained were individually sequenced using the Edman method. The protein sequence is shown in Table 7.1. The light chain is 39 amino acids long and the heavy chain is 91 amino acids long. Why was it necessary to carry out a minimum of two different proteolytic cleavages of the protein using different proteases? One of the enzymes used by the investigators was trypsin. Write the sequences of the fragments that would result from trypsin digestion. Choose a second protease to cleave the light and heavy chains. What protease did you choose, and why? Write the sequences of the fragments that would result from the digestion of the protease you chose.

Solution

Answer:

B. Trypsin cuts at Lys and Arg (at the carboxy terminal side)

Light chain fragments: R, IPK, CR, K, EFQQAQHLR, ACQQWLHK, QANQSGGGPS

Heavy chain fragments: (Please follow the same procedure as the light chain)

C. Chymotrypsin is the protease of choice for a second round of cleavage. Apart from trypsin, chymotrypsin is very efficient in producing reliable cleavage products for sequencing (CNBr can also be a protease of choice).

Chymotrypsin cuts at Phe, Tyr and Trp (at the carboxy terminal side)

Light chain fragments: RIPKCRKEF, QQAQHLRACQQW, LHKQANQSGGGPS

Heavy chain fragments: (Please follow the same procedure as the light chain)