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Biochemistry > Biochem Protein Lecture 4 > Flashcards

Biochem Protein Lecture 4 Flashcards

1
Q

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

A

Purify

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2
Q

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

A

Fractionation

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3
Q

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?

A

solubility

Salts

ammonium sulfate, (NH4)2SO4

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4
Q

Ammonium sulfate has a high or low solubility in water?

A

High

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5
Q

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

Are proteins larger or smaller than their solvent molecules?

A

Dialysis

Larger

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6
Q

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

A

Semi-permeable membrane

Ionic Strength

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7
Q

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.

A

Column Chromatography

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8
Q

The solid matrix packed in the chromatography column is the _____________

The solution flowing through the column is the _______

A

Stationary Phase

Mobile Phase

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9
Q

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

A

Mobile Phase

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10
Q

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

What is the con?

A

Improves resolution of different protein factors

It increases elution time and diffusional spreading within each protein band

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11
Q

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

A

Ion exchange

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12
Q

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

A

Cation Exchange

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13
Q

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

A

Anion Exchange

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14
Q

This type of CC separates proteins according to size.

Do larger or smaller proteins elute first? Why?

A

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

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15
Q

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

A

Affinity CC

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16
Q

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

A

HPLC

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17
Q

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

A

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

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18
Q

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

A

Electrophoresis

19
Q

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

A

Visualized

20
Q

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

A

Electrophoresis

21
Q

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

A

Molecular weight

22
Q

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

A

2

23
Q

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

A

The smaller polypeptides migrate more rapidly

24
Q

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

A

Two-Dimensional Electrophoresis

25
Q

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 ______

A

Isoelectric points (pI)

Molecular Weight

26
Q

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?

A

Hydrolizing

The peptide bond

Strong Acid
Strong Base
Peptidases (enzymes)

27
Q

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?

A

HCl

A mixture of free α‐amino acids

28
Q

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

A

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)

29
Q

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?

A

NaOH

Cys, Ser, Thr, and Arg are destroyed

It can measure Trp content

30
Q

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

A

Low (

31
Q

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

A

Ion exchange; cation-exchange column

32
Q

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

A

Negatively charged resin

Hydrophobic AAs

33
Q

In cation-ion exchange chromatography, the most ___ protein elutes first

The most ____ molecule elutes last

The ______ elute between the above two

A

Acidic

Basic

Hydrophobic AAs

34
Q

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

A

Ninhydrin solution;

Purple-blue compound

Spectrophotometer

35
Q

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

A

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

36
Q

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

A

Polar

37
Q

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?

A

Polar; Nonpolar

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

38
Q

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?

A

Acidic

Basic

39
Q

Does AA composition provide protein structural information?

A

No

40
Q

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?

A

Primary Structure

Peptide and disulfide bonds

41
Q

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

A

Residue

42
Q

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

A

Secondary Structure

43
Q

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

A

Tertiary

Quaternary

44
Q

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

A

Primary

Function

Biochemistry (27 decks)

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