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Chapter 5: Q5-4CP (page 109)
Describe how a protein may be quantified by an assay or by absorbance spectroscopy.
A protein may be quantified by observing the rate of product formation bycoupled enzyme assay, radioimmunoassay (RIA), and enzyme – linked immunosorbent assay (ELISA).
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Question: Sketch a phylogenetic tree for the family of homologous proteins
whose partial sequences are given below.
Protein A T L A D K A I S L H D S
Protein B T L G D K A V S I H E S
Protein C T L A D K A I S V H D S
(a) In what order would the amino acids Arg, His, and Leu be eluted from a carboxymethyl column at pH6 ?
(b) In what order would Glu, Lys, and Val be eluted from a diethylaminoethyl column at pH 8?
You wish to sequence the light chain of a protease inhibitor from the Brassica nigra plant. Cleavage of the light chain by trypsin and chymotrypsin yields the following fragments. What is the sequence of the light chain?
Chymotrypsin
1. Leu–His–Lys–Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser
2. Gln–Gln–Ala–Gln–His–Leu–Arg–Ala–Cys–Gln–Gln–Trp
3. Arg–Ile–Pro–Lys–Cys–Arg–Lys–Phe
Trypsin
4. Arg
5. Ala–Cys–Gln–Gln–Trp–Leu–His–Lys
6. Cys–Arg
7. Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser
8. Phe–Gln–Gln–Ala–Gln–His–Leu–Arg
9. Ile–Pro–Lys
10. Lys
Treatment of a polypeptide with 2-mercaptoethanol yields two polypeptides:
1. Ala–Val–Cys–Arg–Thr–Gly–Cys–Lys–Asn–Phe–Leu
2. Tyr–Lys–Cys–Phe–Arg–His–Thr–Lys–Cys–Ser
Treatment of the intact polypeptide with trypsin yields fragments with the following amino acid compositions:
3. (Ala, Arg, Cys2, Ser, Val)
4. (Arg, Cys2, Gly, Lys, Thr, Phe)
5. (Asn, Leu, Phe)
6. (His, Lys, Thr)
7. (Lys, Tyr)
Indicate the positions of the disulfide bonds in the intact polypeptide.
You wish to determine the sequence of a short peptide. Cleavage with trypsin yields three smaller peptides with the sequences Leu–Glu, Gly–Tyr– Asn–Arg, and Gln–Ala–Phe–Val–Lys. Cleavage with chymotrypsin yields three peptides with the sequences Gln–Ala–Phe, Asn–Arg–Leu–Glu, and Val–Lys–Gly–Tyr. What is the sequence of the intact peptide?
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