The Assay
-Always want to analyze PURE proteins - determine their sequence, function or evolutionary relationship between proteins in diverse organisms
-Proteins can be purified on the basis of differences in their chemical properties
-Assay: a test for some unique
Lactate Dehydrogenase Assay
-Based on the fact that a produce of the reaction (NADH) can be detected spectrophotometrically
-Lactate Dehydrogenase is an enzyme used to convert from lactate to pyruvate
-NADH is strong at 340nm so you check there for the presence of the enzyme
-In ord
Purification
-Presence of a protein you want to extract does not tell you how much of it is present in a sample
-Another critical piece of info: amount of protein present in a mixture being assayed
-As purification proceeds, specific activity rises
-When an enzyme is
Specific Activity
-Information about enzyme activity and protein concentration yields information about specific activity
-Specific activity = units of activity (amount) / amount of total protein
-Want high specific activity
Proteins must be released from the cell to be purified
1. Select a source with high initial specific activity
2. Cells are disrupted to form a homogenate
3. Centrifugation separates nuclei from organelles from soluble proteins (separates soluble from insoluble)
4. Salting out provides partial purification and
Further Purification is Based on...
1. Solubility
2. Size
3. Charge
4. Specific binding activity
-Protein mixtures are subjected to a series of separations, each based on a different property
-At each step, the mixture is assayed, and the amount of wanted protein is determined
-For analysis
Methods of Purification
1. Salting out
2. Dialysis
**Usually done together to remove certain proteins
3. Gel-Filtration Chromatography
4. Ion-exchange Chromatography
5. Affinity Chromatography
6. High-pressure Liquid Chromatography
**Chromatography is used on desired products
Methods of Looking at Protein Characteristics
1. Gel Electrophoresis
-SDS-PAGE
-Isoelectric Focusing
2. Mass Spectrometry
3. X-Ray
4. NMR
**X-Ray and NMR are useful for 3D structures
Salting Out
-Takes advantage of the fact that the solubility of proteins varies with the salt concentration
-Most proteins require some salt to dissolve in water, a process called salting in
-As the salt concentration is increased, different proteins will precipitate
Dialysis
-Salt can be removed from a protein solution by dialysis
-Protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse, but big enough to allow the salt to equilibrate with the solution surrounding the dialysis bag
-
Salting Out/ Dialysis
-Separation based on SOLUBILITY
-Combined to remove salt
General Process of Chromatography
In a container you have beads and these beads will help you interact with certain proteins. Beads can interact by size, charge, or chemical binding affinity. The different characteristics on the beads help to bind to your desired protein and the other pro
3 Main Chromatography Methods
1. Gel Filtration Chromatography
2. Ion-Exchange Chromatography
3. Affinity Chromatography
Gel Filtration Chromatography
-Separation based on SIZE
-Used for isolation
-AKA molecular exclusion chromatography
-A glass column is filled with porous beads and when a protein solution is passed over the beads the large proteins cannot enter the beads so they exit the column first.
Ion-Exchange Chromatography
-Separation by CHARGE
-Beads in the column are made so they have a charge themselves
-When a mixture of proteins is passed through the column, proteins with the same charge as the charge on the beads will exit the column quickly
-Proteins with opposite ch
Ion-Exchange Chromatography - Positive Proteins
Positively charged proteins are separated on carboxymethyl-cellulose (CM-cellulose) columns
Ion-Exchange Chromatography - Negative Proteins
Negatively charged proteins are separated on positively charged diethylaminoethyl-cellulose (DEAE-cellulose) columns
Affinity Chromatography
-Separation based on CHEMICAL BINDING
-Takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups
-Beads are made with specific chemical attacked and a protein mixture is passed through the column; only p
High-Performance Liquid Chromatography
-Using liquid solution (buffer) to make it move
-Much more effective; better resolution
-Using a thin metal column instead of glass
-Time for separation is reduced
-Resolving power of any chromatographic technique is related to the number of potential sit
Purification Summary
-In most cases, several different methods are used sequentially to purify a protein
-For a protein which has not been purified one uses trial and error, literature, data on similar proteins
-Common sense: for the initial mixture, which has the greatest #
Gel Electrophoresis
-Effectiveness of purification step is judged by assessment of specific activity, or by displaying the proteins present at each step
-Electrophoresis is used for the latter - displaying proteins present at each step
-Used as al analytic tool
-Proteins can
2 Types of Gel Electrophoresis
1. SDS-PAGE
2. Isoelectric Focusing
**Each test uses a different gel medium
Gel Electrophoresis Process
-Proteins will migrate in an electric field with a velocity (v) directly proportional to electric field strength (E), the charge on the protein (z) and inversely proportional to the frictional coefficient (f)
**Equation: v = Ez/f
-Frictional coefficient i
SDS-PAGE
-Stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis allows accurate determination of mass
-Medium: vertical slab of polyacrylamide gel
-SDS denatures proteins and for most proteins 1 molecule of SDS binds for every 2 amino acids; thus pr
SDS-PAGE Medium and Dyes
-Gel serving as a molecular sieve that enhances separation
-Usual medium is polyacrylamide inert and readily formed; a 3D mesh due to cross linking
-Small molecules move easily through the sieve than large ones
-Proteins separated by SDS-PAGE are visualiz
SDS-PAGE Mobility
-The mobility is inversely proportional to the log (mass)
-Proteins that differ in mass by 2% (about 10 residues) can be distinguished
-Exceptions: carb rich proteins and membrane proteins do not follow the linear relationship
-Smaller molecules move fast
Purification - Electrophoresis
-Efficacy of purification can be checked by analyzing a part of each fraction by electrophoresis
-The initial fractions will display dozens to hundreds of proteins
-In later stages of purification, the number of bands will diminish and the prominence of o
Isoelectric Focusing
-Less popular than SDS-PAGE
-Based on charge
-Electrophoretic separation based on the relative content of acidic and basic residues in proteins
-If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins wi
Isoelectric Focusing - Process
-pH of the gel changes from one end to the other; there is a pH gradient present in a gel
-Each protein will move until it reaches pH which equals to its pI
-pH gradient is formed by polyampholytes (small multi charged polymers)
2D Electrophoresis
-In 2D gel electrophoresis, proteins are separated in one direction by isoelectric focusing
-This gel is then attached to an SDS-PAGE gel and electrophoresis is performed at a 90 degree angle to the direction of the isoelectric focusing separation
-Horizo
Evaluation of Purification Effectiveness
-At each step of purification, the procedure is monitored by determining specific activity and/or visualization by electrophoresis
-For specific activity, one needs to determine:
1. Total protein (use an assay characteristically of proteins vs other molec
Good vs. Bad Purification
- A good purification scheme should have both high purification levels and high yield
-A high yield and low purification level produce a sample with many contaminants
-A low yield and high purification level produce a sample with an insufficient amount of
Mass Spectrometry
-A powerful technique for identification of peptides and proteins
-For use after obtaining pure protein
-Allows the highly accurate and sensitive detection of the mass of the molecule of interest, or analyte
-Convert the analyte into gas-phase ions; the m
MALDI-TOF Mass Spectrometry
1. The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam
2. An electric field accelerates the ions through the flight tube toward the detector.
3. The lightest ions arrive first.
4. The ionizing laser pulse a
Peptide Sequencing By Mass Spectrometry
-Using mass spectrometry is an alternative to Edman degradation as a means of sequencing proteins
-Amino acid sequence can be determined by Edman degradation
-Protein is exposed to phenyl isothiocyanate (PTH), which reacts with the N-terminal amino acid t
Edman Degradation
-Traditional method to see the amino acid sequence
-Labeling attaches PTH to the sequence and then it releases the end terminal amino acid
-Process repeats and peptide is shortened by one residue
Genomic and Proteomic Methods are Complementary
-Even with today's techniques, determining the amino acid sequence of a protein is somehow a challenge
-Genomic techniques that reveal the DNA sequence of the gene encoding a protein also allow determination of the amino acid sequence
Amino Acid Sequence of a Protein Provides Valuable Info
-Amino acid sequences of proteins can be compared to identify similarities
-Comparison of the same protein from different species yields to evolutionary information
-Amino acid sequence searches can reveal the presence of internal repeats
-Sequencing info
X-Ray Crystallography / 3D Structure
-Gives you most precise 3D structure
-It reveals the position of most atoms in a protein
-Crystals of proteins are irradiated with X-Rays
1. Electrons of the atoms scatter x-rays
2. The scattered waves recombine
3. The way in which the scattered x-rays re
X-Ray Crystallography
-Part of beam of X-rays passes through the crystal and part gets scattered
-Scattered x-rays are detected by a film
-Nature and position of atoms control the scattering, so the scattering pattern contains abundant useful information
-Electrons scatter x-r
X-Ray Diffraction Pattern
Represents electron density cloud; where there is more intensity (darkness) there are more electrons
Integration of Electron Density Map
-A segment of an electron-density map is drawn as a 3D contour plot, in which the regions inside the "cage" represent the regions of highest electron density.
-A model of the protein is built into this map so as to maximize the placement of atoms within t
X-Ray Crystallography - Resolution Effect
-Critical to the interpretation of the map is its resolution, which is determined by the number of scattered intensities used in the Fourier transform
-The fidelity of the image depends on this resolution
-Higher resolution is better
NMR - 3D Structure
-NMR stands for nuclear magnetic resonance
-Reveals atomic structure of macromolecules in a solution
-Solutions must be of sufficiently high concentration
-NMR is based on the fact that certain atomic nuclei are intrinsically magnetic and can exist in two
NMR - Alpha and Beta Spins
-By irradiation the alpha spin can transition to the beta spin and that gives you the NMR spectrum
-Transition between the spin states gives you NMR line
One Dimensional NMR
-Reveals changes to a particular chemical group under different conditions
-Use this to deduce changes to a particular chemical group under different conditions, such as the conformational change of a protein from a disordered structure to an alpha helix
The Nuclear Overhauser Effect (NOE)
-Basis for the NOESY technique
-An interaction between nuclei that is proportional to the inverse sixth power of the distance between them.
-Magnetization is transferred from an excited nucleus to an unexcited one if the two nuclei are less than about 5 �
Nuclear Overhauser Enhancement Spectrometry (NOESY)
-Displays pairs of protons that are in close proximity
-3D structure of a protein is reconstructed form info about the proton pairs the are close to each other
-Structures are calculated with distance constraints based on the info from NOESY spectrum
REVIEW: In x-ray crystallography x-rays are scattered by _______
Electrons
REVIEW: In NMR, one detects _______ which act like magnets
Nuclei
REVIEW: In x-ray crystallography of proteins are in ________
Crystals
REVIEW: In NMR proteins are in ________
Solution
REVIEW: In x-ray crystallography the diffraction pattern created by the x-rays is used to construct an ________
Electron Density Map
REVIEW: In 2D NMR, the experiment gives us ________ that limit the model that can be built or the protein
Restraints