Biochem Protein Lecture 4

Question 1

Biochemists need to isolate and ____ proteins to study their structure and function

Answer 1

Purify

Question 2

To purify proteins from the extract, this technique is used, which involves separating protein into different fractions based upon size or charge

Answer 2

Fractionation

Question 3

Early in the fractionation process, differences in protein _____ are utilized. Proteins can be selectively precipitated from the extract by the addition of certain ____

What is the molecule used for this process?

Answer 3

solubility

Salts

ammonium sulfate, (NH4)2SO4

Question 4

Ammonium sulfate has a high or low solubility in water?

Answer 4

High

Question 5

How is the fractionalized protein separated from ammonium sulfate, the salt?

Are proteins larger or smaller than their solvent molecules?

Answer 5

Dialysis

Larger

Question 6

During dialysis, the protein extract is placed in a tube made of ______

This tube is placed in a larger volume of buffered solution of appropriate _________. Salt and buffer are exchanged through the structure mentioned above

The larger protein molecules stay within the dialysis bag, removing salt from the protein

Answer 6

Semi-permeable membrane

Ionic Strength

Question 7

A widely used method for fractionating proteins, it consists proteins migrating through a column to different extents based upon their interactions with the sold matrix.

Answer 7

Column Chromatography

Question 8

The solid matrix packed in the chromatography column is the _____________

The solution flowing through the column is the _______

Answer 8

Stationary Phase

Mobile Phase

Question 9

In column chromatography, the protein sample is loaded at the top of the column and allowed to diffuse into the solid phase. Additional ______ is added on top causing the proteins to migrate through the column at varying speeds

Answer 9

Mobile Phase

Question 10

What is the pro of increasing column length in column chromatography?

What is the con?

Answer 10

Improves resolution of different protein factors

It increases elution time and diffusional spreading within each protein band

Question 11

This type of CC is based on the interaction of protein with positive or negatively charged chemical groups covalently linked to the polymer beads in the solid matrix

Answer 11

Ion exchange

Question 12

This type of ion exchange CC involves negatively charged (anionic) bound groups, proteins with a net + charge interact with the negatively charged beads and migrate more slowly. Positively charged proteins elute later

Answer 12

Cation Exchange

Question 13

This type of ion exchange CC involves positively charged (cationic) bound groups, so the proteins with a net negative charge interact with the positively charged beads and migrate more slowly. Negatively charged proteins elute later

Answer 13

Anion Exchange

Question 14

This type of CC separates proteins according to size.

Do larger or smaller proteins elute first? Why?

Answer 14

Size-exclusion

Larger proteins elute first because they cannot enter the pores of the polymer beads, as they are too large. They take a shorter path

Question 15

This type of CC is based on protein binding affinity for a chemical group that is covalently attached to the beads in the column. Proteins with affinity to the chemical group bonded to the beads gets help up on the column and elute later

Answer 15

Affinity CC

Question 16

A modern refinement in chromatography that uses high pressure pumps and highly chromatographic materials, resulting in greatly improved resolution

Answer 16

HPLC

Question 17

During HPLC, as a protein is purified, the total protein activity INCREASES/DECREASES while the specific activity INCREASES/DECREASES

Answer 17

During HPLC, as a protein is purified, the total protein activity DECREASES while the specific activity INCREASES

Question 18

This technique is based on the migration of charged particles in an electric field. The basic principles have been described in previous lectures

Answer 18

Electrophoresis

Question 19

Electrophoresis is especially useful as an analytical method because proteins can be ____ as well as separated

Answer 19

Visualized

Question 20

This technique allows for the number of proteins and the purity of a particular protein can to be estimated, as well as the pI

Answer 20

Electrophoresis

Question 21

In SDS gel electrophoresis, the detergent sodium dodecyl sulfate binds to most proteins in amounts prortional to the ____ of the protein

Answer 21

Molecular weight

Question 22

In SDS gel electrophoresis, 1 SDS molecules for every ____ AA residues

Note that the native conformation of the protein is altered when SDS is bound; most proteins assume a similar shape

Answer 22

2

Question 23

In SDS electrophoresis, do the smaller or larger polypeptides migrate more rapidly?

Answer 23

The smaller polypeptides migrate more rapidly

Question 24

This stechnique combines SDS electrophoresis and isoelectric focusing, permitting complex mixtures of proteins to be resolved

Answer 24

Two-Dimensional Electrophoresis

Question 25

In two-dimensional electrophoresis, the first dimension separates proteins by their _____ on a cylindrical gel In the second dimension, the gel is laid horizontally across the top of the polyacrylamide gel and the bands are separated by ______

Answer 25

Isoelectric points (pI) Molecular Weight

Question 26

The amino acid analysis of proteins involves three steps, the first step is ____ing the protein into its constituent amino acids Which bond is cleaved? What is hydrolysis catalyzed by?

Answer 26

Hydrolizing The peptide bond Strong Acid Strong Base Peptidases (enzymes)

Question 27

During hydrolysis catalyzed by a strong acid, protein is dissolved in 6M _______ sealed in an evacuated glass tube and heated at 105-110 degrees celsius for about 24 hours What results?

Answer 27

HCl A mixture of free α‐amino acids

Question 28

Complete hydrolysis is not enough for exact analysis of AA composition due to _____ ______ is almost completely degraded Amide bonds of _____ and _____ are cleaved during acid hydrolysis to produce ____ and ______ + NH4 Small amounts of ___, ____, and _____ are lost

Answer 28

side reactions Tryptophan ``` Asparagine (asn) and glutamine (gln) to make (asp) and glu ``` Ser, Thr, and Tye (aa's containing OH in their side chains)

Question 29

During base hydrolysis, the protein is dissolved in 2-4 M ______ at 100 degrees celsius for 4-8 hours What side reactions occur? What is the benefit of base hydrolysis?

Answer 29

NaOH Cys, Ser, Thr, and Arg are destroyed It can measure Trp content

Question 30

A mix of endo and exopeptidases can be used to completely hydrolyze a protein without changing the AA composition Caution: the concentration of peptidases must be HIGH/LOW because hydrolysis of a peptidase molecule by other peptidase molecules also contributes to the mixture of AAs Benefit: peptidase hydrolysis can determine ___ _____ and ____ content

Answer 30

Low (

Question 31

To separate amino acids in the mixture of hydrolyzed AAs, the separation was done by ____ chromatography on a ____ column

Answer 31

Ion exchange; cation-exchange column

Question 32

A cation resin (sulfonated polystyrene) separates the amino acids in two ways This passes the acidic AA's first and retains the basic ones These get held up by the hydrophobic nature of polystyrene

Answer 32

Negatively charged resin Hydrophobic AAs

Question 33

In cation-ion exchange chromatography, the most ___ protein elutes first The most ____ molecule elutes last The ______ elute between the above two

Answer 33

Acidic Basic Hydrophobic AAs

Question 34

The third step of determining composition of AAs is to quantitate it as it elutes from the column In automated AA analysis, the effluent is mixed with a ______ solution, which reacts with AA and ammonia to form a ___________ compound The colored solution is passed through a flow cell of a _______; the absorbance is proportional to the concentration of the AA The amount of each AA is determined from the area under each peak of the absorbance vs. time elution curve

Answer 34

Ninhydrin solution; Purple-blue compound Spectrophotometer

Question 35

More recently, AAs are derivatized with o‐phthalalaldehyde and β‐ mercaptoethanol and separated by _____ Separation is based on differences in solubility of AA derivatives in a POLAR/NONPOLAR solvent relative to POLAR/NONPOLAR The resin has POLAR/NONPOLAR groups on its surface, POLAR/NONPOLAR solvent molecules surround it and form a POLAR/NONPOLAR stationary phase

Answer 35

reverse‐phase chromatography Separation is based on differences in solubility of AA derivatives in a NONPOLAR solvent relative to POLAR solvent The resin has NONPOLAR groups on its surface, NONPOLAR solvent molecules surround it and form a NONPOLAR stationary phase

Question 36

In reverse phase chromatography, the mobile phase is POLAR/NONPOLAR

Answer 36

Polar

Question 37

In reverse phase chromatography, the ___ derivatives elute first, while the ___ derivative elute last The AAs can be quantized by UV absorption, refractive index or fluorescence Which AA elutes last? Why?

Answer 37

Polar; Nonpolar Lysine because both the α‐amino and the ε‐amino groups are derivatized, making the lysine derivative the least polar

Question 38

The presence of AAs in any given proteins is variable. The AA composition provides information on the pI of the protein If protein has more acidic AA, the pI is in which range? If the protein has more basic AA, the pI is in which range?

Answer 38

Acidic Basic

Question 39

Does AA composition provide protein structural information?

Answer 39

No

Question 40

This structure of proteins is the sequence of AA residues and description of all covalent bonds linking aa residues in a polypeptide chain What are mainly the bond type in this structure?

Answer 40

Primary Structure Peptide and disulfide bonds

Question 41

This term refers to what remains of an amino acid after formation of peptide bonds

Answer 41

Residue

Question 42

This structure of proteins is particularly stable arrangements of AA residues, with recurring structural patterns (like an alpha helix)

Answer 42

Secondary Structure

Question 43

This protein structure is all aspects of 3D folding of a polypeptide chain This protein structure type is assembled subunits, the arrangement of 2 or more polypeptides in space

Answer 43

Tertiary Quaternary

Question 44

The ______ structure of proteins determines how it fold into its unique 3D structure; which in turn determines protein _____

Answer 44

Primary Function