Biochem Protein Lecture 4 Flashcards
Biochemists need to isolate and ____ proteins to study their structure and function
Purify
To purify proteins from the extract, this technique is used, which involves separating protein into different fractions based upon size or charge
Fractionation
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?
solubility
Salts
ammonium sulfate, (NH4)2SO4
Ammonium sulfate has a high or low solubility in water?
High
How is the fractionalized protein separated from ammonium sulfate, the salt?
Are proteins larger or smaller than their solvent molecules?
Dialysis
Larger
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
Semi-permeable membrane
Ionic Strength
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.
Column Chromatography
The solid matrix packed in the chromatography column is the _____________
The solution flowing through the column is the _______
Stationary Phase
Mobile Phase
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
Mobile Phase
What is the pro of increasing column length in column chromatography?
What is the con?
Improves resolution of different protein factors
It increases elution time and diffusional spreading within each protein band
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
Ion exchange
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
Cation Exchange
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
Anion Exchange
This type of CC separates proteins according to size.
Do larger or smaller proteins elute first? Why?
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
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
Affinity CC
A modern refinement in chromatography that uses high pressure pumps and highly chromatographic materials, resulting in greatly improved resolution
HPLC
During HPLC, as a protein is purified, the total protein activity INCREASES/DECREASES while the specific activity INCREASES/DECREASES
During HPLC, as a protein is purified, the total protein activity DECREASES while the specific activity INCREASES
This technique is based on the migration of charged particles in an electric field. The basic principles have been described in previous lectures
Electrophoresis
Electrophoresis is especially useful as an analytical method because proteins can be ____ as well as separated
Visualized
This technique allows for the number of proteins and the purity of a particular protein can to be estimated, as well as the pI
Electrophoresis
In SDS gel electrophoresis, the detergent sodium dodecyl sulfate binds to most proteins in amounts prortional to the ____ of the protein
Molecular weight
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
2
In SDS electrophoresis, do the smaller or larger polypeptides migrate more rapidly?
The smaller polypeptides migrate more rapidly
This stechnique combines SDS electrophoresis and isoelectric focusing, permitting complex mixtures of proteins to be resolved
Two-Dimensional Electrophoresis
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 ______
Isoelectric points (pI)
Molecular Weight
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?
Hydrolizing
The peptide bond
Strong Acid
Strong Base
Peptidases (enzymes)
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?
HCl
A mixture of free α‐amino acids
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
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)
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?
NaOH
Cys, Ser, Thr, and Arg are destroyed
It can measure Trp content
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
Low (
To separate amino acids in the mixture of hydrolyzed AAs, the separation was done by ____ chromatography on a ____ column
Ion exchange; cation-exchange column
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
Negatively charged resin
Hydrophobic AAs
In cation-ion exchange chromatography, the most ___ protein elutes first
The most ____ molecule elutes last
The ______ elute between the above two
Acidic
Basic
Hydrophobic AAs
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
Ninhydrin solution;
Purple-blue compound
Spectrophotometer
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
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
In reverse phase chromatography, the mobile phase is POLAR/NONPOLAR
Polar
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?
Polar; Nonpolar
Lysine because both the α‐amino and the ε‐amino groups are derivatized, making the lysine derivative the least polar
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?
Acidic
Basic
Does AA composition provide protein structural information?
No
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?
Primary Structure
Peptide and disulfide bonds
This term refers to what remains of an amino acid after formation of peptide bonds
Residue
This structure of proteins is particularly stable arrangements of AA residues, with recurring structural patterns (like an alpha helix)
Secondary Structure
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
Tertiary
Quaternary
The ______ structure of proteins determines how it fold into its unique 3D structure; which in turn determines protein _____
Primary
Function
