Determination Of A.a

Question 1

How to determine a.a composition

Answer 1

Protein-hydrolysed(acid/alkali/enzyme-liberate a.a-separated by chromatography
Common used enzyme-pronase-cmplte hydrolysis
Enzyme-results in smaller peptides

Question 2

To degrade protein into smaller fragments

Answer 2

Liberation of polypeptide
No of polypeptide
Breakdown into fragments

Question 3

Liberation of polypeptide

Answer 3

Urea or guanidine hcl-non covalent bonds into polypeptide
Performic acid-cleaving the disulfide linkage

Question 4

No of polypeptide

Answer 4

Dansyl chloride with protein binds to N terminal-dansyl polypeptide-n terminal acid-dansyl a.a
No ofdansyl a.a =no of polypeptide chain

Question 5

Breakdown into fragments

Answer 5

Enzymatic-proteolytic enzymes (trypsin-common,chymotrypsin,pepsin,elastase)or
OR
chemical method -cyanogen bromide (CNBr)
Split polypeptide into fragments

Question 6

To determine a.a sequence

Answer 6

Sangers reagent-FDNB(1 fluro 2,4 dinitrobenzene)-binds to n terminal a.a-DNP (dinitrophenyl)-hydrolysis-DNP a.a +free a.a
DNP-identified by chromatography

Edmans reagent-phenyl isothiocyanate-react with n-terminal -phenyl carbomyl +mild acid-phenyl thiohydantoin(PTH)
Identified by chromatography

Sequentor-automatic machine
Principle of Edmans degradation
From N-terminal PTH Identified by HPLC(high performance liquid chromatography)
2 hrs to determine each a.a

Question 7

Eg of primary structure

Answer 7

Human insulin

Question 8

2 precussors of insulin(51a.a)

Answer 8

Preproinsulin(108a.a)
Proinsulin(86 a.a)

Question 9

Formation of insulin

Answer 9

Preproinsulin-leaving signal sequence-proinsulin-insulin+c-peptide

Question 10

Two chains A and B

Answer 10

Chain A(21 a.a)
Chain B(30 a.a)

Question 11

Disulphide chains in insulin

Answer 11

Interchain-A7 to B7 and A19 to B20
Intrachain on chain A-A6 and A11