bioc 205 exam 2

concerns of stabilizing proteins during purification

pH: -requires the use of a buffer to maintain the pH in a range where the protein will not denature temperature: -thermal stability of proteins varies. many proteins denature at high temperatures. therefore most purifications are carried out at between 0-4°C. note that the air-liquid interface is a prime spot for denaturation degradative enzymes: -these can be proteases or nucleases. these can be "controlled" by pH, temperature, and inhibitors adsorption to surfaces: -most proteins bind readily. the fewer steps the better!

rate law

reaction rate: r=k[A][B] A= reactant 1 B= reactant 2 C=product k= rate constant r= reaction rate

steps of assays

1. develop assay 2. get access to your protein in the starting material 3. purify

how mass spectrometry works

1. ionize 2. accelerate 3. deflect 4. detect

considerations of protein purification

1. what is the intended use of the final product? 2. what's my starting material? 3. what are the properties of my protein? 4. what is my monitoring system/assay?

2D gel electrophoresis

1st dimension: IEF 2nd dimension: SDS-PAGE results in a collection of protein spots analytical technique

protein fragmentation strategies

3 agents can be used to produce an overlapping set of peptides if each peptide is determined, the entire protein sequence can be reassembled

protein structure

3D representation of a protein

antibody structure

4 individual polypeptide chains (2 heavy, 2 light) connected by disulfide bonds variable regions bind epitopes, specifically through 3 complementary-determining regions (CDRs) that make up the antigen binding site constant regions serve remaining functions, such as recombination by immune cells the Fc (c for crystallizable) region is the "stem" portion made up of portion of the heavy chains the Fab region (ab for antigen-binding) region contains the portions of the heavy and light chains with the antigen binding site

identifying an unknown protein

AAA, protein sequencing, mass spectrometry

why don't antibodies simply bind to everything in our bodies

B cells making antibodies against our own proteins are killed off

how are 100,000,000 different Igs possible when we only have ~30,000 genes

random recombination of Ig gene segments take place

k₁

rate constant of forward direction r=k₁[A][B]

k₋₁

rate constant of reverse direction r=k₋₁[P][Q]

size exclusion (gel filtration) chromatography

a glass column is filled with porous beads. when a protein solution is passed over the beads, large proteins can't enter the beads and exit the column first. small proteins can enter the beads and thus have a longer path and exit the column last can be used in both preparative and analytical ways, acts as a preparative technique for this class

centrifugation

a homogenate is formed by disrupting the cell membrane centrifugation is a method for fractionation -the supernatant is the liquid portion -the pellet contains the "denser," "heavier," "larger," "insoluble" material can be used to separate particles based on degrees of size and insolubility but also on the basis of density via density gradient centrifugation (isopycnic centrifugation). in the latter case, a density gradient of a solution (such as CsCl or sucrose) is prepared in the centrifugation tube, and solutes migrate to positions based on their own density preparative technique

enzyme linked ImmunoSorbent Assay (ELISA)

a label on the antigen is an enzyme that produced a chemical reaction when interacting with another substance. enzyme activity is directly related to the concentration of antigen present analytical technique

amino acid analysis (AAA)

a method for determining the identity and quantity of constituent amino acids in a protein protein is boiled in HCl for 24+ hours until all the peptide bonds have been hydrolyzed separation by HPLC (either ion-exchange or reversed-phase) samples are treated with ninhydrin or other reagents to enable spectrophotometric detection Asn and Gln are converted to Asp and Glu respectively Trp is destroyed B: Asp+Asn Z: Glu+Gln

principles of column chromatography

a porous resin (solid phase) with certain chemical properties is held in a glass cylinder -a buffered solution (mobile phase) percolates through -protein solution is applied in the mobile phase and percolates through as an expanding band -different proteins migrate differently depending on their properties and those of the resin can be used in both preparative and analytical ways, acts as a preparative technique for this class

homogenization

a preparative technique A homogenized sample is equal in composition throughout, so that removing a fraction does not alter the overall molecular make-up of the sample remaining, and is identical to the fraction removed

dialysis

a procedure for exchanging the solvent around a protein the protein solution is placed inside a semi-permeable membrane (dialysis bag) which is suspended in a larger volume of buffered solution the membrane is permeable to water and ions, but not to proteins buffers and salts exchange until an equilibrium is established between the inside and outside of the membrane preparative technique

Fab region of antibody

ab for antigen-binding contains the portions of the heavy and light chains with the antigen binding site

purifying proteins based on binding affinity

affinity chromatography

antibody

allow vertebrates to fend off invading pathogens through an immune response immunoglobulin proteins recognize foreign substances (antigens)

mass spectrometry

an analytical technique that produces spectra of the masses of the atoms or molecules comprising a sample of material is NOT mass spectroscopy either ion source type can work with either mass analyzer type (ex: MALDI-TOF, ESI-Quad)

cell-mediated immunity

an immune response that doesn't involve antibodies, but rather involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen

assay

an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology, and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity (the analysis)

hydrophobic interaction chromatography (HIC)

at high ionic strength, proteins are partially desolvated, causing them to adopt alternate conformations in which normally buried hydrophobic residues are more exposed. these residues can then form hydrophobic interactions with the hydrophobic functional groups conjugated to a matrix. lowering the ionic strength causes the protein to refold into it's native conformation, burying it's hydrophobic residues. this decreases hydrophobic interactions between the protein and stationary phase, facilitating protein elution can be used in both preparative and analytical ways, acts as a preparative technique for this class

NMR structure

atomic model based on the interpretation of a set of interatomic distances. a "solution" structure pros: native protein in solution, a "family" of structures with potential to see molecular movement cons: must be stable at high concentration, size limit of 30-40 kDa 9,583 models of proteins (<300 residues) (compared to 74,131 models for x-ray crystallography)

x-ray crystallography structure

atomic model based on the interpretation of high resolution x-ray crystal diffraction data pros: no practical size limits, don't need to maintain high concentration of protein in solution cons: protein must crystallize, only one snapshot of protein 74,131 models of proteins and nucleic acids (compared to 9,583 models for NMR)

affinity chromatography

beads are made with the specific chemical attached a protein mixture is passed through the column only proteins with affinity for the attached group will be retained the bound protein is then released by passing a solution enriched in the chemical to which the protein is bound can be used in both preparative and analytical ways, acts as a preparative technique for this class

enzymes

biological catalysts accelerate chemical reactions under physiological (mild) conditions most are proteins great reaction specificity capacity for regulation not changed or used up after a reaction nomenclature: add "-ase"

Fc region of antibody

c for crystallizable the "stem" portion made up of portion of the heavy chains

getting access to your protein in the starting material in an assay

cells (bacteria, yeast, mammalian), tissue, plants, etc. are all composed of many proteins first step in the purification process usually involves making a crude extract by breaking the cells open to release their contents inhibitors, buffers, etc. are added to hinder decomposition, denaturation initially large cellular material is separated by filtration or light centrifugation

charge of proteins and ion exchange chromatography

changed by the pH of the solution -low pH: proteins are positively charged → column material in chromatography is negatively charged cation exchanger -pI: the pH at which the net charge is 0 -high pH: proteins are negatively charged → column material in chromatography is positively charged anion exchanger

hydroxylamine at pH 9 protein fragmentation

cleaves Asn-Gly bonds

pH 2.5 40°C protein fragmentation

cleaves Asp-Pro bonds

trypsin

cleaves protein after Arg and Lys

staph protease

cleaves protein after Asp and Glu

cyanogen bromide (CNBr)

cleaves proteins after Met

chymotrypsin

cleaves proteins after Tyr, Phe, Trp

carboxypeptidase A

cleaves successive AAs from the C-term of peptides

aminopeptidase

cleaves successive AAs from the N-term of peptides

chemical protein fragmentation

cyanogen bromide (CNBr) hydroxylamine at pH 9 pH 2.5 40°C

detection in mass spectrometry

depending on the intensity of the magnetic field applied, only ions of a certain m/z will pass through the mass analyzer and reach the detection unit at the back of the device. ions with larger and smaller m/z values hit the sides of the spectrometer and are neutralized. to analyze all the ions, chemists adjust the intensity of the magnetic field until each stream hits the detector

reverse-phase chromatography

done via HPLC (high performance liquid chromatography) is a similar concept to HIC in which samples are loaded in a fairly hydrophilic mobile phase, so hydrophobic molecules bind to the column due to the hydrophobic effect, and then a more hydrophobic mobile phase is added, solubilizing those hydrophobic molecules and allowing them to elute. C18 (18-carbon alkyl chain) is a very common chemical group used in reverse phase HPLC can be used in both preparative and analytical ways, acts as a preparative technique for this class

types of ion sources in mass spectrometry

electrospray (ESI) matrix assisted laser desorption ionization (MALDI)

titration curves of proteins

entire proteins have titration curves

B lymphocytes (B cells)

expresses membrane-bound immunoglobulins on their surface capable of recognizing virtually any chemical structure each B cell expresses just one specific type of immunoglobulin on its surface, but at any time there are roughly 100,000,000 different types of immunoglobulins present in the body these immunoglobulins are similar throughout much of their structure, except for their antigen binding variable region (shown in image) when a membrane-bound immunoglobulin on the surface of a B cell binds to a foreign antigen, it signals for that cell to proliferate and secrete soluble version of that immunoglobulin known as antibodies wherever the antigen is found in the body, these now plentiful antibodies will bind to it, which marks it for destruction by other immune cells (such as phagocytes that will engulf the antigen and the pathogen on which it's found)

order of chemical reactions

for a simple unimolecular reaction such as A→P, the rate law is r=k[A] for a bimolecular reaction such as A+B→P+Q, the rate law is r=k[A][B] for an elementary reaction, the "order" of any reactant is given by it's exponent in the rate equation the top reaction (A→P) is a unimolecular reaction, and is 1st order with respect to A and 1st order overall the bottom reaction(A+B→P+Q) is a bimolecular reaction, and is 1st order with respect to A and B, and second order overall knowing "order" is helpful in studying reactions (fitting curves mathematically, simplifying them, etc)

antigen

foreign substance identified by antibodies

purifying proteins based on size

gel filtration chromatography

purifying proteins based on stability

heat treatment

when considering starting material in protein purification

historically, abundance and easy isolation dictated which proteins were isolated and therefore studied (e.g. hemoglobin) many proteins are common to all species (e.g. metabolic enzymes) and therefore they could be isolated from various sources (e.g. yeast, bovine) and used in model systems molecular biology has made it possible to isolate anything, regardless of it's natural abundance

kinetics

how fast will the reaction happen? an enzyme can make it go faster by reducing the activation energy "hump"

purifying proteins based on hydrophobicity

hydrophobic interaction chromatography

polyclonal antibodies

if one injects an antigen into an animal (from another organism since if the animal's tolerance is working, it won't have antibodies to it's own structures) that animal will go through the process of B cell proliferation and antibody production the animal will make multitudes of any of it's antibodies that recognize the antigen, and one can obtain some of the animal's blood and purify those freshly made antibodies chances are, even if only a single protein from another organism was injected in to the animal, numerous specific types of antibodies will have been made because there are numerous molecular structures called "antigens" on various parts of that protein this collection of various antibodies recognizing one protein (or other macromolecule) is called a polyclonal antibody because the many "poly" antibodies in the collection came from many unique B cells or "clones" not as specific as monoclonal antibodies because the mixture of antibodies (from various lines of B cells) recognizes a collection of epitopes on the protein of interest

monoclonal antibodies

in monoclonal antibody technology, antibody producing B cells taken from the spleen of a mouse, rabbit, etc are combined with immortal tumor cells that can replicate endlessly the result of this cell fusion is a "hybridoma" hybridoma cells are diluted into cell culture Wells so that each well contains ~1 cell, the individual cells are allowed to proliferate and secrete their unique antibody these antibodies are called monoclonal because they come from only one "clone," one type of hybridoma cell (derived from 1 type of B cell); monoclonal antibodies are highly specific - they recognize just one epitope if the monoclonal isolation procedure isn't done, the mixture of antibodies is called polyclonal monoclonal is more specific than polyclonal

salting out

initial steps in a purification procedure often utilize the differences in protein solubility, which is a complex function of pH, temperature, salt concentration, composition, etc solubility of proteins is generally lower at high salt concentrations, leading to a "salting out" effect. specific control of the concentration can selectively participate some and leave others in solution ammonium sulfate (NH₄)₂SO₄ is most often used for salting out because of it's low price, great availability in pure form, and high solubility, which allows for solutions of very high ionic strength molecular basis involves the salt interacting with so many of the water molecules in the solution that there aren't enough to support interactions with proteins and keep them soluble those proteins that have more interactions with water (i.e. are more soluble) can withstand higher salt concentrations, and can be separated from protein that have fewer interactions with water preparative technique

purifying proteins based on charge

ion-exchange chromatography

result of mass spectrometry

mass spectrum as the stream of ions passes through the mass spectrometer, the magnetic field deflecting the ions is adjusted to scan and sequentially allow a range of m/z particles through. the detector records how many ions hit it at the various intensities of the magnetic field, and you get a mass spectrum

when considering properties of protein in protein purification

molecular size pI solubility (hydrophobicity) sensitivity/stability with respect to: -pH -detergents -tempetature -metal ions -salt concentration -organic solvents -redox state -need for cofactors sample complexity (PAGE) contaminants

developing assays

most assays are chemical reactions catalyzed by specific enzymatic activities proteins that have no activity are usually assayed using SDS polyacrylamide gels (PAGE) or with antibodies since the assay is repeated many times, it is important that is be a simple procedure many different forms: -spectroscopic (bradford, enzyme activity (chromogenic substrate))

visualization of proteins in protein purification

most frequently Coomassie Brilliant Blue stain is used gels can be transferred to nitrocellulose (western blot) and probed with specific antibodies western blotting and visualizing via PAGE are analytical techniques

column chromatography

most powerful of the fractionation methods separates components of a mixture based on: -size -charge -hydrophobicity -binding affinity can be used in both preparative and analytical ways, acts as a preparative technique for this class

ion exchange chromatography

negatively charged beads bind and elute positively charged (cationic) proteins positively charged beads binds and elute negatively (anionic) proteins high salt - can disrupt ionic interactions an H-bonding cation exchanger: negative charged beads anion exchanger: positively charged beads can be used in both preparative and analytical ways, acts as a preparative technique for this class

protein sequencing

once you have obtained a PURE protein, it can be sequenced intact proteins may have blocked N-termini, disulfide bonds, etc. making characterization difficult "divide and conquer" strategy is normally employed - fragment the protein into peptides that can easily be analyzed

why we want to see protein structures

rational drug design hypothesis testing

immune response

pathogenic organism in body → antigens (proteins, etc) from organism bind to specific B cells through unique secretory surface immunoglobulin (sIg) on the cell surface → in those B cells binding antigen, a signal for activation and proliferation is induced → clonal expansion produces numerous copies of the cell → cells undergo differentiation into plasma cells, producing and secreting large quantities of the specific immunoglobulin that got the process started → the Fab portion of the specific antibody binds to the original antigen on the pathogen, the resulting conformational change allows the Fc portion of the antibody to be bound by the Fc receptor on immune cells, and the pathogen is destroyed by being engulfed (phagocytes) or lysed (other white blood cells)

crude extracts

protein from cells or tissue a crude extract is not the "natural" environment of the protein of interest, therefore it may be exposed to many agents that could irreversibly damage it!

sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE)

protein mixtures are denatured and coated with SDS (negatively charged) disulfides are typically broken with β-mercaptoethanol all proteins assume a rod like shape with a uniform charge/mass ratio thus, all secondary and tertiary and quaternary structure is lost analytical technique

immunoglobulin (Ig)

protein with antibody activity

when considering monitoring system/assay in protein purification

proteins are sub-microscopic and usually colorless how will you know where your protein is throughout all your numerous purification steps? assays are analytical and typically involved techniques such as spectrophotometry, enzyme-catalyzed reaction, etc

Polyacrylamide gel electrophoresis (PAGE)

proteins vary widely in their charge, size, and shape electrophoretic mobility of small molecules is greater than the mobility of large molecules with the same charge density pH of the buffer and protein mixture is high (~9) so that the proteins carry a net-negative charge an electric field is used to separate the proteins molecules of similar size and charge move through the gel as a band without detergent (SDS), the proteins retain their normal "native" shape so the technique is called "native PAGE" without SDS: protein shape and charge do matter in the separation

x-ray crystallography and NMR

provide complementary experimental data

when considering intended use of final product in protein purification

purity varies extremely high purity (>99%) has therapeutic use and in vivo studies high purity (95-99%) used in x-ray crystallography and most physico-chemical characterization methods moderate purity (< 95%) used as antigen for antibody production and in N-terminal sequencing

types of mass analyzers in mass spectrometry

quadrupole (Quad, Q) ion trap time-of-flight (TOF) analyzers can be combined to create "hybrid" instruments (ex: ESI-QQQ, MALDI QQ TOF, Q Trap)

hybridoma

result of monoclonal antibody technology an immortalized cell which will continually produce antibodies

purifying proteins based on solubility

salting out

electrophoresis

separates components of a mixture based upon their charge and/or size -paper electrophoresis -polyacrylamide gel electrophoresis (PAGE) -SDS-PAGE -isoelectric focusing (IEF) analytical technique

purification of proteins

separation methods will make use of physical properties/differences of proteins all methods used for purifying proteins are preparative steps

humoral immunity

the aspect of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides named so because it involves substances found in the humors, or body fluids

deflection in mass spectrometry

the mass analyzer applies an external field that interacts with the magnetic field generated by the fast-moving particles, causing the path of each particle to bend slightly. how much an ion's path curves depends on 2 factors: the mass of the ion and its charge. lighter ions and ions with a greater charge are deflected more than heavier ions and ions with a smaller charge

spectrophotometry

the only really reliable method for protein concentration measurements. the molar extinction coefficient of a protein at 280 nm can be calculated with reasonable accuracy from it's amino acid sequence as the sum of the contributions of all tryptophans, tyrosines, and disulfide bridges analytical technique

relating equilibrium constants and rate constants

the rate at which a reaction proceeds is dictated by the concentrations of the chemical components, and the rate constant (the natural inherent rate at which a specific reaction happens to occur, which is determined by experiments) the overall rate of the reaction is the product of these elements: r=k[A][B] multiplying these components together to get the rate makes sense because: 1. the more of the chemical components (A and B) that are present, the more frequently they can collide and the faster they will react 2. the faster the inherent rate of that reaction (k) is, the faster they will react all chemicals reactions are in reality reversible, so that helps understand equilibrium at equilibrium reverse rate is equal to forward rate

chemical kinetics and enzyme kinetics

the rate is proportional to the concentration of A, and it is also proportional to the rate constant, k, which like an equilibrium, K, is a constant based on the inherent properties of the chemicals themselves

epitope

the region of the antigen that is recognized by an antibody

spectroscopy

the study of the interaction between matter and radiated energy NOT mass spectrometry

analytical techniques

those used to identify and quantify your protein - they often destroy your sample, but you are only using a small part of the material at each step of the purification process

preparative techniques

those used to manipulate and purify your protein for future use - they are going to be gentle to your protein of interest because you're going to continue using it

acceleration in mass spectrometry

to move the ions into the mass spectrometer, the force required comes from an electric field supplied by 2 metal grids. 1 grid is positively charged and repels the ions, the other is negatively charged and attracts them. because the repulsion and attraction act in the same direction, the ions move rapidly toward the negatively charged grid, which is perforated with many tiny holes

proteases protein fragmentation

trypsin chymotrypsin staph protease carboxypeptidase A aminopeptidase

edman degradation

type of protein sequencing 1. N terminal AA reacts with phenylisothiocyanate 2. derivatized AA is cleaved in acid 3. derivatized AA is released as the phenylthohydantoin (PTH) derivative 4. derivatized AA is identified by AAA 5. released derivatized AA reveals the next AA for another round of sequencing

equilibrium constant

uppercase "K"s are equilibrium constants, they deal with with the equilibrium/thermodynamics of a reaction and often have units of concentration (mM etc) lowercase "k"s are rate constants, they deal with the rate (kinetics) of a reaction and have units of time (per second, minute) in chem, biochem, etc. big K's (equilibrium, association, dissociation constants) are made up of small k's (rate constants)

western blotting

used to detect specific protein molecules from among a mixture of proteins starts with SDS-PAGE (which denatures the proteins)

paper electrophoresis

used to separate AAs or peptides of differing charge anions travel toward anode cations travel toward cathode analytical technique

how we see protein structures

using NMR or x-ray crystallographic techniques

ionization in mass spectrometry

very common technique for ionizing samples (such as those coming from chromatographic separation) is electrospray ionization. forcing samples in solution through a narrow (< 10 µm) opening with high voltage applied converts the solute into charged particles in the gas phase

isoelectrofocusing (IEF)

when the pH<pI, the protein moves to the cathode (-) when the pH>pI, the protein moves to the anode (+) when the pH=pI, the protein will not move analytical technique

thermodynamics

will the reaction happen? the available energy in a system which can be converted into work at a constant temperature and pressue reactions will proceed (as the reaction is written down left to right) if the ∆G (change in Gibbs free energy) is negative ∆G=∆H-T∆S A+B→C+D negative is favorable positive is unfavorable enzymes do not change the equilibrium of a reaction, only accelerate the reaction by decreasing the activation energy