BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

SECTION I. CONFORMATION AND DYNAMICS

QUESTIONS AND PROBLEMS

1. The following Reagents are commonly used in Protein Chemistry:

CNBr Dansyl chloride

6 N HCl β-Mercaptoethanol

Urea Trypsin

Ninhydrin Phenylisothiocyanate

Perperformic acid Chymotrypsin

Which of these reagents should be used to solve the following problems:

a) determining the Amino Acid Sequence of a small peptide;

б) identification of the amino-terminal residue in a peptide (in an amount of less than 10^-7 g);

c) reversible Denaturation of a protein lacking Disulfide Bonds. What additional reagent would be required if the protein contains disulfide bonds?

d) Hydrolysis of peptide bonds at the carboxyl group of aromatic amino acid residues;

e) Cleavage of peptide bonds at the carboxyl group of methionines;

f) hydrolysis of peptide bonds at the carboxyl groups of Lysine and Arginine residues?

2. What is the pH of the following solutions:

a) 10^-3 N HCl;

б) 10^-2 N NaOH;

c) a mixture of equal volumes of 0.1 M acetic acid and 0.03 M sodium acetate;

d) a mixture of equal volumes of 0.1 M Glycine and 0.05 M NaOH;

e) a mixture of equal volumes of 0.1 M glycine and 0.05 M HCl?

3. What is The ratio of base to acid at pH 4, 5, 6, 7, and 8, given that the pK of the acid is 6?

4. The Muscle protein Tropomyosin is a coiled-coil consisting of two α-helical strands. The Molecular Weight of this protein is 70 kDa. The average molecular weight of a single amino acid residue is about 110 Da. Calculate the length of the molecule.

5. Anhydrous hydrazine is used to cleave peptide bonds in Proteins. What are the reaction products? How could this method be utilized to identify the C-terminal amino acid?

6. Human adrenocorticotropic hormone is a polypeptide with the following amino acid sequence: Ser-Tyr-Ser-Met-Glu-

His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys- Lys-Arg-Arg-Pro-V al-Lys-Val-T yr-Pro-Asp- Ala-Gly-Glu-Asp-Gln-Ser-Ala-Glu-Ala-Phe- Pro-Leu-Glu-Phe.

a) Estimate the overall net charge of this molecule at pH 7, assuming that the pK values of The amino acid side chains are those given in Table 2.3, and that the pK values of the terminal —NH₃⁺ and —COOH groups are 7.8 and 3.6, respectively.

b) How many Peptides will be obtained upon Treatment of the hormone with Cyanogen bromide?

7. Ethylenimine reacts with Cysteine side chains in a protein to yield S-aminoethyl derivatives. Peptide bonds formed by the carboxyl groups of these modified cysteine residues are cleaved by trypsin. Why?

8. An enzyme catalyzing exchange reactions between disulfide and sulfhydryl groups has been isolated. Inactive "scrambled" Ribonuclease is rapidly converted into its catalytically active form by the action of this enzyme. On the other hand, this same enzyme is rapidly inactivated by Insulin. What Conclusion regarding the relationship between the amino acid sequence of insulin and its three-dimensional Structure can be drawn from this observation?