о но N. NH NH2 DNP-1 o-N DNP-2 IZ
Determine the sequence of the following 10-mer of ferritin using the data below. i. Complete acid hydrolysis of the 10-mer results in identification of 1A, 1D, 1E, 1F, 1H, 1K, 2L, 1T, 1Y ii. Digestion of the 10-mer with chymotrypsin results in 3 fragments containing (D, H, K, T), (F, L) and (A, E, L, Y) iii. Digestion of the 10-mer with V8 protease, which cuts on the C-side of acidic amino acids, results in three fragments containing (D, F, L, Y), (A, E, L) and (H, K, T) iv. Digestion with trypsin results in a fragment H-T and a fragment with all the other amino acids v. Treatment of the 10-mer with FDNB followed by complete acid hydrolysis results in the following two derivatives, DNP-1 and DNP-2 vi. Treatment of the 10-mer with a carboxypeptidase results in a free threonine
Acid hydrolysis: Acid hydrolysis of amino acids hydrolyze the peptide bond and gives the amino acid sequence of any polypeptide chain. Here acid hydrolysis gives us details of all the amino acids present in the given polypeptide they are A (alanine), D (aspartic acid), E(glutamic acid), F(phenylalanine), H(histidine), K (lysine), L(leucine), T(threonine) and Y(tyrosine).
Chhymotrypsine digestion : Chymotrypsine cleaves the C-terminal of aromatic amino acids i.e., C-terminal of F & T amino acids in the given sample.
V-8 protease digestion: V-8 protease is a serine endopeptidase that use to cleave the carboxylic side of E (glutamic acid) & D (aspartic acid). Therefore it can be inferred that D and E aren't terminal amino acids in the given polypeptide chain as digestion generates three polypeptide chains.
Trypsin digestion: Proteolysis targets of trypsin into polypeptide chain are carboxylic side of K (lysine) or R (arginine). Trypsin digestion of the given polypeptide generates two polypeptides (one containing H, T, and the other containing K and others) and only K is present in the amino acid sequence. From this, it can be concluded that K is at the 3rd position from the C-terminal.
FDNB treatment: FDNB uses to label the N-terminal of any polypeptide chain. Here FDNA treatment followed by acid proteolysis generates DNP-1 and DNP-2 thus L (leucine) must be present at the N-terminal of the polypeptide chain.
Carboxypeptidase digestion: It uses to cleave C-terminus of any polypetide chain.
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