Lista 3 -

Poznawanie białek (c.d.)

- 1 z 2-

B

IOCHEMIA

I

–

ĆWICZENIA

LISTA 3

do wykładu dr. hab. inż. P. Dobryszyckiego

P o z n a w a n i e b i a ł e k ( c . d . )

1. Which of the following statements concerning the Edman degradation method are true?

(a) Phenyl isothiocyanate is coupled to the amino-terminal residue.
(b) Under mildly acidic conditions, the modified peptide is cleaved into a cyclic derivative of the

terminal amino acids and a shortened peptide (minus the first amino acid).

(c) Once the PTH amino acid is separated from the original peptide, a new cycle of sequential

degradation can begin.

(d) If a protein has a blocked amino-terminal residue (as does N-formyl methionine, for example), it

cannot react with phenyl isothiocyanate.

2. Which of the following are useful in identifying the amino-terminal residue of a protein?

(a) cyanogen bromide

(d)

dabsyl chloride

(b) fluorodinitrobenzene

(e)

phenyl isothiocyanate

(c) performic acid

3. What treatments could you apply to the following hemoglobin fragment to determine the amino-

terminal residue and to obtain two sets of peptides with overlaps so that the complete amino acid
sequence can be established? Give the sequences of the peptides obtained.

Val-Leu-Ser-Pro-Ala-Lys-Thr-Asn-Val-Lys-Ala-Ala-Trp-Gly-Lys-Val-
Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Thr-Glu

4. Which of the following techniques are used to locate disulfide bonds in a protein?

(a) The protein is first reduced and carboxymethylated.
(b) The protein is cleaved by acid hydrolysis.
(c) The protein is specifically cleaved under conditions that keep the disulfide bonds intact.
(d) The peptides are separated by SDS-polyacrylamide gel electrophoresis.
(e) The peptides are separated by two-dimensional electrophoresis with an intervening performic

acid treatment.

5. Match the terms in the left column with the appropriate item or items from the right column.

(a) antigens

(1)

immunoglobulins

(b) antigenic determinants

(2)

foreign proteins, polysaccharides, or nucleic acids

(c) polyclonal antibodies

(3)

antibodies produced by hybridoma cells

(d) monoclonal antibodies

(4)

groups recognized by antibodies

(5)

heterogeneous antibodies

(6)

homogeneous antibodies

(7)

antibodies produced by injecting an animal with a
foreign substance

(8)

epitopes

6. The methods used to localize a specific protein in an intact cell are

(a) Western blotting

(d) immunoelectron microscopy

(b) solid-phase immunoassay

(e) fluorescence microscopy

(c) enzyme-linked immunosorbent assay

Lista 3 -

Poznawanie białek (c.d.)

- 2 z 2-

7. Explain why immunoassays are especially useful for detecting and quantifying small amounts of a

substance in a complex mixture.

8. The following reagents are often used in protein chemistry:

(a) CNBr
(b) urea
(c) β-mercaptoethanol
(d) trypsin
(e) dicyclohexylcarbodiimide
(f) dabsyl chloride
(g) 6 N HCl
(h) fluorescamine
(i) phenyl isothiocyanate
(j) chymotrypsin
(k) dilute F

3

CCOOH

Which of these reagents are best suited for the following tasks?

1) determination of the amino acid sequence of a small peptide
2) identification of the amino-terminal residue of a peptide (of which you have less than 10

-7

grams)

3) reversible denaturation of a protein devoid of disulfide bonds
4) hydrolysis of peptide bonds on the carboxyl side of aromatic residues
5) cleavage of peptide bonds on the carboxyl side of methionine
6) hydrolysis of peptide bonds on the carboxyl side of lysine and arginine residues

7)

reversible denaturation of a protein that contains disulfide bonds (two reagents are

needed)

8) activation of carboxyl groups during peptide synthesis
9) determination of the amino acid composition of a small peptide

10)

removal of t-Boc protecting group during peptide synthesis

9. A peptide composed of 12 amino acids does not react with dabsyl chloride or with phenyl

isothiocyanate. Cleavage with cyanogen bromide yields a peptide with a carboxyl-terminal
homoserine lactone residue, which is readily hydrolyzed, in turn yielding a peptide whose
sequence is determined by the Edman procedure to be

E-H-F-W-D-D-G-G-A-V-L

On the other hand, cleavage with staphylococcal protease (see Table 4.3 in text) yields an
equivalent of aspartate and two peptides. Use of the Edman procedure gives the following
sequences for these peptides:

G-G-A-V-L-M-E and H-F-W-D

Why does the untreated peptide fail to react with dabsyl chloride or phenyl isothiocyanate?