Protein Sequencing

Question 1

5 Steps in determining the primary structure.

Answer 1

1. Identify the amino acid present and their proportions. (Hydrolyzation)
2. Determine the N- and C-terminal amino acids in protein sequence.
3. Cleave the protein in smaller fragments and determine the amino acid.
4. Repeat step 3 using a cleavage procedure at different specific sites
5. Determine the complete amino acid sequence.

Question 2

EXPLANATION: Identify the amino acid present and their proportion (hydrolyzation.)

Answer 2

Acid hydrolysis leads to destruction of W, S, C, T. Using 6M HCl at 100-110 degree celsius, 12-36hrs.

Base hydrolysis leads to racemization. Using 4N NaOH at 100 degree celsius, 10hrs.

This hydrolysis helps to separate the identity of the individual amino acids. To detect them we use Amino acid analyzer and HPLC (High performance liquid chromatography)

Question 3

EXPLANATION: Determine the N- and C-terminal amino acid.

Answer 3

It can check the presence of one or more polypeptides chain by using Edman method. This will help to locate where the N- and C-terminal is.

Question 4

EXPLANATION: Cleavage of the protein.

Answer 4

Cleave the protein in small fragments and determine the sequence of amino acids sequence. It determine as much of sequence as possible.

In cleavage we use different enzymes to cleave the fragments. And where amino acids they cleave.

Then, we combine the two step in cleavage by overlapping the peptides to get the complete sequence.

Question 5

Cleavage:

In internal, what enzyme cleaves the carboxyl side of positively charge R group? Amino acids are Arg and Lys.

Answer 5

Trypsin

Question 6

Cleavage:

In internal, what enzyme cleaves the amino side of aromatic amino acid? Amino acids are Tyr, Trp, Phe and Leu

Answer 6

Pepsin

Question 7

Cleavage:

In internal, what enzyme cleaves the carboxyl side of aromatic amino acid? Amino acids are Tyr, Trp, and Phe.

Answer 7

Chymotrypsin

Question 8

Cleavage:

In terminal, What enzyme use to cleaves amino side of C-terminal amino acid?

Answer 8

Carboxypeptidase

Question 9

Cleavage:

In terminal, What enzyme use to cleaves carboxyl side of N-terminal amino acid?

Answer 9

Aminopeptidase

Question 10

Cleavage:

In internal, it cleaves carboxyl side of Methionine?

Answer 10

Cyanogen bromide

Question 11

Cleavage:

Terminal, uses 2,4-dinitrofluorobenzene and form DNP derivative with N-terminal amino acid?

Answer 11

Sanger’s Method

Question 12

Cleavage:

It forms PTH (phenythiohydantion) derivative with N-terminal aminod acid?

Answer 12

Edman Degradation

Question 13

Cleavage:

It hydrolyzes all peptide bonds and releases free C-terminal amino acids?

Answer 13

Hydrazinolysis

Question 14

Cleavage:

Forms a sulfonamide derivative with N-terminal amino acid?

Answer 14

Danzyl Chloride Treatment

Question 15

Aminopeptidase

Ala-Phe-Pro-Val-Gly-Leu

Answer 15

Ala + Phe-Pro-Val-Gly-Leu

Question 16

Carboxypeptidase

Ala-Phe-Pro-Val-Gly-Leu

Answer 16

Ala-Phe-Pro-Val-Gly + Leu

Question 17

Chymotrypsin

Ala-Phe-Arg-Val-Gly-Leu-Lys-Pro

Answer 17

Ala-Phe + Arg-Val-Gly-Leu-Lys-Pro

Question 18

Trypsin

Ala-Phe-Arg-Val-Gly-Leu-Lys-Pro

Answer 18

Ala-Phe-Arg + Val-Gly-Leu-Lys + Pro

Question 19

Pepsin

Ala-Phe-Arg-Val-Gly-Leu-Lys-Pro

Answer 19

Ala + Phe-Arg-Val-Gly + Leu-Lys-Pro

Question 20

Sanger’s Method

Ala-Phe-Arg-Val-Gly-Leu-Lys-Pro

Answer 20

DNP-Ala + Phe-Arg-Val-Gly-Leu-Lys-Pro

Question 21

Cyanogen bromide

Ala-Phe-Arg-Val-Met-Leu-Lys-Pro

Answer 21

Ala-Phe-Arg-Val-Met + Leu-Lys-Pro