chapter 5 section 3

Question 1

What does acid hydrolysis do to a protein?

Answer 1

it breaks proteins into individual amino acids.

Question 2

What is a limitation of acid hydrolysis?

Answer 2

Some amino acids are partially or completely destroyed.

Question 3

How are amino acids separated after hydrolysis?

Answer 3

Using chromatographic methods.

Question 4

Do different proteins have the same amino acid composition?

Answer 4

No, each protein has a unique amino acid composition.

Question 5

What does the primary structure of a protein refer to?

Answer 5

The specific sequence of amino acids in the polypeptide chain.

Question 6

What methods are used to determine protein sequences?

Answer 6

Chemical, enzymatic, and physical methods.

Question 7

What are the two main approaches to protein sequencing?

Answer 7

Direct amino acid sequencing and sequencing the corresponding DNA.

Question 7

Who first sequenced a protein and what was it?

Answer 7

frederick Sanger sequenced insulin in 1953.

Question 8

What did Sanger’s sequencing show?

Answer 8

All molecules of a given protein have the same sequence.

Question 9

What is special about insulin’s structure

Answer 9

It has two polypeptide chains (A and B) connected by disulfide bridges.

Question 10

How many amino acids are in the A and B chains of bovine insulin?

Answer 10

A chain has 21 residues; B chain has 30 residues.

Question 11

What is the first step in the six-step protein sequencing strategy?

Answer 11

Separate and purify polypeptide chains.

Question 12

How are subunits separated?

Answer 12

Using extreme pH or high salt concentrations.

Question 13

What is step 2 in protein sequencing?

Answer 13

Cleaving disulfide (S-S) bridges.

Question 14

Name two methods to cleave disulfide bonds.

Answer 14

Performic acid oxidation and reduction with sulfhydryl agents

Question 15

Why are alkylating agents used after S-S reduction?

Answer 15

To prevent reformation of disulfide bonds.

Question 16

What is step 3 of sequencing?

Answer 16

Identify the N-terminal and C-terminal residues

Question 17

How is the N-terminal identified?

Answer 17

Using Edman degradation with phenylisothiocyanate (PTH derivatives).

Question 18

Which residues does carboxypeptidase A not cleave?

Answer 18

Proline, arginine, and lysine.

Question 19

How is the C-terminal identified?

Answer 19

Using enzymatic analysis with carboxypeptidases.

Question 20

Which residues does carboxypeptidase B cleave?

Answer 20

Only arginine and lysine.

Question 20

What is step 4 in protein sequencing?

Answer 20

Cleaving each polypeptide into smaller fragments.

Question 21

Name two types of fragmentation methods.

Answer 21

Enzymatic and chemical fragmentation.

Question 22

Which enzyme cleaves at the C-side of phenylalanine, tyrosine, and tryptophan?

Answer 22

Chymotrypsin.

Question 22

Which enzyme cleaves at the C-side of lysine and arginine?

Answer 22

Trypsin.

Question 23

Which enzyme prefers arginine over lysine?

Answer 23

Clostripain.

Question 24

Which residues does staphylococcal protease cleave in phosphate buffer

Answer 24

Glutamate and aspartate.

Question 25

Which residue does cyanogen bromide cleave?

Answer 25

Methionine.

Question 26

What is produced at the C-terminal after CNBr cleavage?

Answer 26

Homoserine lactone residues.

Question 27

What is step 5 in sequencing?

Answer 27

Repeating fragmentation with a different agent to generate overlapping fragments.

Question 28

What is step 6 in sequencing?

Answer 28

Reconstruct the full amino acid sequence from overlapping fragments.

Question 29

What does mass spectrometry separate proteins by?

Answer 29

Mass-to-charge ratio (m/z).

Question 29

What physical method can also determine amino acid sequences?

Answer 29

Mass spectrometry (MS).

Question 30

How are protein fragments generated for MS?

Answer 30

Enzymatic digestion, chemical cleavage, or other fragmentation techniques.

Question 31

What is tandem mass spectrometry (MS/MS) used for?

Answer 31

Sequencing peptides by measuring mass differences between fragments.

Question 31

How is the amino acid sequence determined from tandem MS?

Answer 31

By calculating mass differences corresponding to individual residues.