This document discusses various techniques for determining the primary, secondary, tertiary, and quaternary structures of proteins. It describes methods such as determining amino acid composition, degradation of proteins into smaller fragments, sequencing techniques like Edman degradation, and use of X-ray crystallography and NMR to analyze secondary and tertiary structures. Chromatography, electrophoresis, and centrifugation techniques are also covered for protein purification and separation.

2. Contents
Determination of primary structure
Determination of secondary and tertiary
protein structures

3. Determination of primary structure
Determination of aminoacid composition
Degradation of protein or polypeptide into smaller fragments.
Determination of the aminoacid sequence

4. Determination of amino acid composition in a
protein
1. acid or
2. alkali treatment
3. enzyme hydrolysis Pronase
Separation and estimation of amino acids: chromatography

5. Degradation of protein into smaller fragment
liberation of polypeptides
urea or
guanidine hydrochloride
performic acid

6. Which of the following is a denaturing substance
A Guanosine
B Guanidine
C Glutamate
D Glycine

7. Breakdown of polypeptides into fragments
trypsin, chymotrypsin, pepsin and elastaseEnzymatic cleavage:
Chemical cleavage: Cyanogen bromide

8. Number of polypeptides
dansyl chloride.
binds N-terminal aminoAcids form dansyl polypeptide
on hydrolysis yield N-terminal dansyl amino acid

9. Determination of amino acid sequence

10. 1-fluoro-2,4-dinitrobenzene (FDNB)
binds with N-terminal amino acids to form Dinitrophenyl (DNP)
derivative of peptide
• This is on hydrolysis yields DNP – amino acids ( N-terminal) and
free amino acids from the rest of the peptide chain.
• Used for identification of N terminal AA
• DNP-AA – identified by Chromatography
Sanger's reagent

11. Phenyl isothiocyanate
Reacts with N-terminal amino acid
Phenyl thiohydantoin (PTH)-amino acid is liberated
Identified by chromatography
Edmans reagent

12. Useful in sequencing first 10-30 amino acids

14. Sequenator
• This is an automatic machine to determine the amino acid
sequence in a polypeptide
• It is based on the principle of Edmans degradation .
• Amino acids are determined sequentially from N-terminal end
• The PTH-amino acid liberated is identified by HPLC.
• Sequenator takes about 2 hours to determine each amino acid.

15. Reverse sequencing technique
• DNA determines the sequence of amino acids in a polypeptide chain
• By analyzing nucleotide sequence of DNA that codes for protein,
translate nucleotide sequence into amino acid sequence.
• Demerits - fails to identify the disulfide bonds
changes that occur in the amino acids after the protein is synthesized.

16. 1. X-ray crystallography
2. Nuclear magnetic resonance (NMR) spectra of proteins
Determination of secondary , tertiary , quarternary protein
structures

19. Electrophoresis

21. Proteins are separated on the basis OF SIZE BY
A.) SDS-PAGE
B.) HPLC
C) Affinity Chromatography
D) Ion -exchange Chromatography
(Pgi June03)

22. MOLECULAR WEIGHT OF A PROTEIN CAN BE DETERMINED BY
A POLY ACRYLAMIDE GEL ELECTROPHORESIS PAGE
B SODIUM DODECYL SULPHATE PAGE
C ISO ELECTRIC FOCUSSING
D ION EXCHANGE CHROMATOGRAPHY

23. Protein purification and separation can be done by all
except
A. Chromatography
B. Centrifugation
C.Electrophoresis
D. Densitometry

24. Chromatography Centrifugation
DIFFUSION

25. IN CHROMATOGRAPHY MASS MOVEMENTS OF THE SUBSTANCE
IS DUE TO
A DIFFUSION
B ELECTROPHORESIS
C PAPER CHROMATOGRPHY
D OSMOSIS

26. Haemoglobin electrophoresis

27. In the case of Sickle Cell hemoglobin ,
replacement of a negatively-charged Glu in the
standard HbA beta-globin by a
neutral Val in HbS
results in a protein with a slightly reduced negative charge.

28. Hemoglobin electrophoresis is based on
A molecular weight
B Charge..
C Solubility
D calorimetric properties

29. gel filtration / diffusion chromatography
BEST METHOD
Proteins
Nucleic acids

30. Cellulose resins have much greater permeability to
macromolecular polyelectrolytes
Carboxymethyl cellulose (CM-cellulose) – Cationic exchanger
DEAE cellulose - Anionic exchanger

33. Cationic exchangers possess negatively charged group,
and these will attract positively charged cations.
“Acidic ion exchange materials”,
because their negative charges result from the ionization
of acidic group.

34. Anion exchanger

35. MOLECULAR SEPERATION OF TWO PROTEINS WITH
SAME CHARGE CAN BE DONE BY
A ION EXCHANGE CHROMATOGRAPHY
B DIALYSIS
C GEL DIFFUSION CHROMATOGRAPHY
D ELECTROPHORESIS

36. The following separation technique depends on molecular
size of the protein
A . Chromatography on carboxymethyl (CM) cellulose column
B. Iso-electric focussing
C. Gel filtration chromatography/ exclusion chromatography
D.Chromatography on di ethyI amino ethyl (DEAE) cellulose
column

38. Hydrophobic chromatography
Affinity chromatography
Gel chromatography
Paper chromatography
In -which type of chromatography, proteins are Bound to
another substance

39. X ray diffraction

40. The tertiary structure of protein is detected by:
A. X- ray diffraction
B Spectrophotometry
C.Electrophoresis.
D.Chromatography

41. The substance present in the gall bladder stones or the kidney
A flurosence spectroscopy
B.Electron microscopy
C.Nuclear magnetic resonance
D.X ray diffraction

42. Mass spectroscopy

46. Molecular size is assessed by
A Sedimentation
B Absorption Mass spectroscopy
C.Liophilization
D.Salting out

47. Chromosome walking

48. Sequence in a Iong Chain of protein is identified by-
A. restriction fragment length polymorphism
B Chromosome walking
C. Leucine Zipper
D. SSOP sequence specific oligonucleotide probes