Biochemistry Test 1
Terms
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- BIOMOLECULES
- an organic compound normally present as an essential component of living organisms
- DISTINGUISHING FACTORS OF LIVING ORGANISMS
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- A high degree of chemical complexity and microscopic organization
- Systems for extracting, transforming, and using energy from the environment
- A capacity for precise self-replication and self-assembly
- Mechanisms for sensing and responding to alterations in their surroundings
- Defined functions for each of their components and regulated interaction among them - BIOCHEMISTRY
- - describes in molecular terms the structures, mechanisms, and chemical processes shared by all organisms and provides organizing priciples we refer to collectively as the molecular logic of life
- PLASMA MEMBRANE
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- defines the periphery of the cell
- composed of lipid and protein molecules that form a thin, tough, pliable, hydrophobic barrier around the cell
- free barrier to inorganic ions and most other charged and polar molecules
- allows change of shape and size of cell because of flexibility - CYTOPLASM
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- composed of the cytosol
- the cytosol is highly concentrated with enzymes and RNA molecules, amino acids and nucleotides, metabolites, coenzymes, and ribosomes - NUCLEUS
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- where DNA is stored and replicated
- Eukaryotes have nuclear envelopes while prokaryotes do not - KINGDOMS
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- eubacteria
- eukaryotes
- archaebacteria - EUBACTERIA
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- inhabits soils, surface waters, and the tissue of other living or decay organisms
- Most well studied bacteria - ARCHAEBACTERIA
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-most recently discovered
- less well characterized biochemically
- inhabit extreme environments (salt lakes, hot springs, highly acidic bogs, and ocean depths
- more closely related to eukaryotes than eubacteria - PHOTOTROPHS
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- trap and use sunlight
- autotrophs- receive all needed carbon from CO2
- heterotrophs- need carbon from organic molecules - CHEMOTROPHS
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- energy from oxidation of a fuel (organic nutrients)
- They can't fix carbon dioxide into organic compounds
- can't get carbon exclusively from carbon dioxide
- Lithotrophs- fuels that are oxidized are inorganic
- Organotrophs- fuels that are oxidized are organic - DIFFERENCES BETWEEN EUKARYOTES AND PROKARYOTES
- - eukaryotes are bigger
- DIFFERENCES BETWEEN PLANT AND ANIMAL CELLS
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- animal cells have a nuclear envelope
- plant cells have chloroplasts and vacuoles
- plant cell have a cell wall - CELL FRACTIONATION
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- cells or tissues in solution are disrupted by gentle homogenization
- leaves organelles in tact but ruptures plasma membrane
- homogenate is centrifuged
- organells sediment at different rates because of size - DIFFERENTIAL CENTRIFUGATION
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- rough fractionation of the cytoplasmic contents
- organelles of different buoyant densities are separated on a density gradient - CYTOSKELETON
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- protein filaments crisscrossing the eukaryotic cell
- forms n interlocking three-dimensional meshwork
- 3 types of filament: microtubules, actin, and intermediate filaments
- provide structure and shape to cytoplasm and shape to cell
- interactions between cytoskeleton and organelles are noncovalent, reversible, and subject to regulation in response to variousintracellular and extracellular signals - MACROMOLECULES
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- subunits are usually joined by covalent bonds
- in supramolecular complexes macromolecules are held together by noncovalent interactions - METABOLOME
- - the entire collection of small molecules in a given cell
- PROTEINS
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- most abundant biological macromolecule occurring in all cells and all parts of cells
- molecular instruments through which genetic info is expressed
- polymers of amino acids that are bounded by covalent bonds. Loses water when aa join together (amino acid residue) - NUCLEIC ACIDS
- - nucleotide residues are linked into DNA and RNA
- POLYSACCHARIDES
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- polymers of simple sugars
- energy yielding fuel stores
- extracellular structural elements with specific binding sites for particular proteins - LIPIDS
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- hydrocarbon derivatives
- structural components of membranes
- energy rich fuel stores
- pigments
- intacellular signals - STEREOISOMERS
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- molecules with the same chemical bonds but different stereochemistry
- exists when there is one double bond and has chiral centers
- a molecule has 2^n steroisomers. n being the number of chiral centers - CHIRAL CENTERS
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- carbon with 4 different substituents
- clockwise rotation is R, counterclockwise rotation is S
- a molecule with a single chiral center can be named with the R-S naming system or the D-L naming system - ENANTIOMERS
- - mirror images of each other
- DIASTEREOMERS
- - not mirror images of each other
- RACEMIC MIXTURE
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- 2 enantiomers that exhibit no optical rotation because the same amount of enantiomer exists in the mixture
- Compounds without chiral centers do not rotate the plane of polarized light - CONFORMATIONS
- - staggered is most stable, while eclipsed is least stable
- SYSTEM
- - all reactants and products present, the solvent that contaisn them, and the immediate atmosphere
- CLOSED
- - if it exchanges both energy and matter with its surroundings
- OPEN
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- if it exchanges both energy and matter with its surroundings
- living organism - ISOLATED
- - if the system exchnages neither matter nor energy with its surroundings
- OXIDATION REDUCTION REACTIONS
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- all reactions involving electron flow
- one reactant is oxidized while the other is reduced - ENTROPY
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- change in S
- any change in randomness of the system
- positive when randomness increases - FREE ENERGY CONTENT
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- G
= change H - [T * Change S]
- if G is negative it is a spontaneous reaction - ENTHALPY
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- H
- reflects the number and kinds of bonds - ENDERGONIC REACTIONS
- - energy requiring
- EXERGONIC REACTIONS
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- energy releasing
- the sum of G1 and G2 is negative
- decline in free energy from reactants to products
- ATP to ADP and Pi - Keq
- - the reaction proceeds until the reactants have almost completely converted into the products
- STANDARD FREE ENERGY CHANGE
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- change G = -RT ln Keq
- J/mol
- negative when Keq>>1 - CATABOLIC
- - exergonic reactions
- ANABOLIC
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- endergonic reactions
- requires the input of energy - WATER
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- most abundant substance in living systems (70% or more weight of living systems)
- Important physical and chemical properties
- Solvent for bioreactions
- bond angle is 104.5
- Tetrahedron
- When water is a liquid is bonds to less water molecules (3.4) than when it is with ice (4.0) - WEAK INTERACTIONS IN AQUEOUS SOLUTION
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- hydrogen bonds
- ionic interactions
- hydrophobic interactions
- Van der Waals Interactions - HYDROGEN BONDS
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- gives water its unusual propterties: high melting point, high boiling point, high heat of vaporization
- the electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another
- occurs between an electronegative atom (hydrogen acceptor) like oxygen or nitrogen with a lone pair of electrons and a hydrogen atom covalently bonded to another electonegative atom (hydrogen donor) - BOND DISSOCIATION ENERGY
- - energy required to break a bond
- SPONTANEITY
- - Change of H (enthalpy) remains the same for melting and evaporation, so the increase in entropy (change in S) makes the change of G positive or negative
- WHEN ARE HYDROGEN BONDS THE STRONGEST?
- - molecules are oriented to maximize electrostatic interaction when the hydrogen atom and the two other atoms are in a straight line.
- HYDROPHILIC
- - compounds that dissolve easily in water
- HYDROPHOBIC
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- don't dissolve in water
- nonpolar molecules like lipids and waxes - FORCE OF IONIC INTERACTIONS
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- magnitude of charges (Q1&Q2) divided by the distance and dielectric constant
- stronger in less polar environments - NONPOLAR COMPOUNDS
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- unable to undergo energetically favorable interactions with water molecules
- small gain in enthalpy
- decrease in entropy - AMPHIPATHIC COMPOUNDS
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- contains regions that are polar and regions that are nonpolar
- forces that hold nonpolar areas together are hydrophobic interactions - DISPERSION OF LIPIDS IN WATER
- - each lipid molecule forces surrounding water molecules to become highly ordered
- CLUSTERS OF LIPID MOLECULES
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- only lipid portions at the edge of the cluster force the ordering of water
- fewer water molecules are ordered
- entropy increases - MICELLES
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- circle of lipids
- hydrophobic groups are sequestered from water
- ordered shell of water molecules is minimized
Entropy increases - VAN DER WAALS INTERACTIONS
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- any two atoms in close proximity
- dipoles from electrons near both nucleuses attract one another - COLLIGATIVE PROPERTIES
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- depend only on the solute concentration, not the chemical properties of the solute
- vapor pressure
- boiling point
- melting point
- osmotic pressure
- solutes alter colligative properties by lowering the effective concentration of water - OSMOTIC PRESSURE
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- water molecules diffuse from an area of high concentration to an area of low concentration
- PI = icRT
- c is the molar concentration, I is the van't Hoff factor, R (0.0821 liter-atm/mole degree K) is the gas constant, and T is the absolute temperature
- Pi is given in atmospheres - ISOTONIC
- - no net water movement
- HYPERTONIC
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- water moves out and cell shrinks
- inside the cell has a smaller concentration of molecules than outside the cell - HYPOTONIC
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- water moves in to the higher concentration
- creates outward pressure
- cell swells and eventually bursts - IONIC PROPERTIES OF WATER
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- pure water is slightly ionized
- water = H+ + OH-
- H exists as a hydronium ion which gives up a proton in proton hopping
- very rapid - EQUILIBRIUM CONSTANT
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K = [A][B]/[C][D]
- water is: k= [H][OH]/[water]= 1.8 X 10^-16M - ION PRODUCT OF WATER
- Kw = (55.5M)(k) = [H][OH] = 1.0 x 10^-14 M^2
- pH
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- the ion product of water forms the basis for the pH scale
- -log[H]
- pOH is -log[OH] - STRONG ACIDS
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- completely ionized
- hydrochloric, sulfuric, and nitric
- pH = -log[acid] - STRONG BASES
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- completely ionized
- NaOH and KOH
- pH = 14-log[base] - WEAK ACIDS
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- don't completely dissociate in water
- acids give up protons
- acetic acid - WEAK BASES
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- don't completely dissociate in water
- bases accept protons
- ammonia - CONJUGATE ACID-BASE PAIR
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- HA = H+ + A-
- HA is the conjugate acid
- A is the conjugate base
- Ka = [H][A]/HA - pKa
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= log (1/ka) = -log Ka
= to the midpoint of the titration curve
- measure of the tendency of a group to give up a proton
- the molecules with the lowest pk loses its protons first - TITRATION CURVE OF WEAK ACID
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- a plot of PH against the amount of NaOH added
- a weak acid and a conjucate acid-base pair acts as a buffer
- involves the gradual addition and removal of protons - HENDERSON-HASSELBALCH EQUATION
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Ka = [H][A]/[HA]
logKa = log[H] + log[A]/[HA]
-log[H] = -logKa + log[A]/[HA]
pH = pK + log[A]/[HA] - BUFFERS
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- resist change in pH
- mixtures of weak acids and their conjugate bases resist change in pH upon addition of either stron acid or base
- A buffering region is along the plateau (when the weak acid is 100% titrated) - BUFFERING IN BIOLOGICAL SYSTEMS
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- phosphate and bicarbonate buffer systems maintain intracellular and extracellular fluids at their optimum pH
- optimum pH is close to 7
- Enzymes generally work at optimum pH - CONDENSATION REACTION
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- water is eliminated
- ADp + Pi = ATP
- endergonic reactions - HYDROLYSIS REACTION
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- addition of water elements
- responsible for enzymatic depolymerization of proteins, cabs, and nucleic acids
- exergonic reactions - AMINO ACIDS
- - organic compounds that contain both an amino group and a carboxylic acid
- STEROISOMERS
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- The alpha carbon is the chiral center of the amino acids as long as there are 4 different groups
- amino acids have new steroisomers because they are nonsuperimposible mirror images (enantiomers) and optically active - D,L SYSTEM
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- Its L if the amino group is on the left
- Its D if the amino group is on the right
- Amino acid residues in proteins are L stereoisomers - POLARITY
- - tendency to interact with water at biological pH near 7.0
- NONPOLAR, ALIPHATIC R GROUPS
- - tends to cluster within proteins, stabilizing protein structure by means of hydrophobic interactions
- AROMATIC R GROUPS
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- relatively nonpolar
- all can participate in hydrophobic interactions - POLAR UNCHARGED R GROUPS
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- more soluble in water (hydrophilic)
- functional groups form hydrogen bonds with water - CYSTINE
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- covalently linked dimeric amino acid
- 2 cysteine molecules or residues are joined by a disulfide bond
- disulfide bonds are nonpolar and hydrophobic - POSITIVELY CHARGED (basic)R GROUPS AND NEGATIVELY CHARGED (Acidic) R GROUPS
- - most hydrophillic R groups are either positively or negatively charged
- ZWITTERION
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- dipolar ion
- can act as a acid or base
- also called ampholytes because of this duel nature - ISOELECTRIC POINT
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- at sufficiently low pH all amino acids are positively charged
- At sufficiently high pH all amino acids are negatively charged
- At some pH all amino acids have no charge
- The isoelectric point is the pH at which the amino acid has no net charge
- pI = 1/2(pk1 + pk2)
* Must be the two pks that surround the molecule with no net charge - DIFFERENCES IN PKs
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- The basic diprotic titration is exhibited in the amino acids with a single alpha amino group and a single alpha carboxyl group, and an R group that doesn't ionize
- The normal range is pk (COOH) 1.8 to 2.4 and pK (NH3) 8.8 to 11.0
- Amino acids with a ionizable R group have a 3 stage titration curve - PEPTIDES
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- polymers of amino acids
- peptide bond is the only bond
- written with amino terminus at left and carboxyterminus at right
- aa are called residues - PEPTIDE BOND
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- covalently bonds 2 amino acids with a substituted amide linkage
- removes water
- joins alpha carboxyl group of one aa with the alpha amino group of the other
- hydrolysis is exergonci but its slow because of high activation energy
- peptide bonds are stable and have a half life of 7 years
- characteristic titration curve and isolectric pH - OLIGOPEPTIDE
- - few amino acids are joined
- POLYPEPTIDE
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- many amino acids are joined together
- molecular weight below 10,000. Proteins have molecular weight above 10,000 - ISOELECTRIC PH FOR PEPTIDES
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- determined by loss of charge in alpha carboxyl and alpha amino groups, the interactions with other peptide R groups, and environmental factors
- approximate number of amin o acid residues by dividing its molecular weight by 110. - MULTISUBUNIT
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- 2 or more polypeptide chains bonded noncovalently
- If they are identical polypeptides the protein is oligomeric and the units are protomers - SIMPLE PROTEINS
- - only amino acid residues and no other chemical constituents
- CONJUGATED PROTEINS
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- proteins with permanently associated chemical ponents in addition to amino acids
- classified based on prosthetic groups - PROSTHETIC GROUP
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- non amino acid part of the conjugated protein
- lipoproteins, glycoproteins, metalloproteins - PROTEIN STRUCTURE
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- Primary - amino acid sequence and includes any disulfide bonding that exists
Covalent bonds between amino acids and proteins
- Secondary- stable arrangements
- Tertiary- 3 dimensional folding of a polypeptide
- Quarternary- two or more polypeptide subunits
- protomers are the smallest units - PROTEIN PURIFICATION
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- Assay (know how much protein your starting with by enzymatic activity, immunological activity, color, size or any property you can measure)
- breaks open tissue and microbial cells and releases it into crude extract
- Separate - differential centrifugation can be performed to prepare subcellular fractions or isolate specific organelles
- purify proteins through fractionation (separated on size and charge) The best way to do this is by column chromatography
- combine fractions with the protein
- Repeat with a new separation Method - DETERMINING AMINO ACID COMPOSITION FROM PEPTIDE
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- hydrolyze peptide to free amino acids
- separate mino acids from acid hydrolysis or base hydrolysis (use other methods to determine Q, N, W, and C
- quantitate amino acids through spectrophotometric method
A = e[c]l
A is absorbance, c molar concentration, and e is molar extinction (object absorbed light if incident light is greater than the transmitted light - AMINO ACID COMPOSITION
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- composition of peptide or protein is number of moles of each amino acids present in one mole of the peptide
- only whole numbers because its the number of each aa/ # of protein - DETERMINING CYSTEINE
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- oxidize with performic acit to cysteic acid
Alkylate with iodoacetate or iodoacetamid to make carboxymethylcysteine or carboxamidomethylcysteine
- these are stable for acid hydrolysis - DTT
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- helps understand protein
- Reduce disulfide bond to form free disulfidal groups
- only works in basic conditions because sulfhydral group needs to be a nucleophile
- intramolecular reaction
- only molecule that reacts with iodoacetate - SIZE-EXCLUSION CHROMATOGRAPHY
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- separates proteins according to size
- the smaller the protein then it has more volume accessible to it since it can get inside the tube easier.
- Bigger proteins go in first and the smaller ones go in last. - CATION-EXCHANGE CHROMATOGRAPHY
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- separation of protein based on charge
- Fill the column with a solid matrix of either positve or negative charged molecules.
- If the column has negatively charged molecules the positive charge proteins migrate through the matriz more slowly
- Resolution improves when the column is longer - TYPES OF FUNCTIONAL GROUPS ON COLUMNS
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- amino group (NH3+)
- Carboxyl group (-COO-)
- Sulfonic acid (SO3-)
- Phosphate Groups (PO42- - HYDROPHOBIC INTERACTION CHROMATOGRAPHY
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- hydrophobic groups on column interact with hydrophobic surfaces on protein
- interaction is favored by high salt concentration
- Sample applied in high salt, and then the salt concentration is steadily decreased
- Proteins elute in decreasing order of hydrophobicity - AFFINITY CHROMATOGRAPHY
- - beads in the column have covalently attached chemical groups. A protein with this affinity will attach to that particular group and its migration will stop.
- ACTIVITY
- - total units of enzyme in a solution
- SPECIFIC ACTIVITY
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- the number of enzyme units per milligram of total protein
- a measure of enzyme purity - SDS POLYACRYLAMIDE GEL ELECTROPHORESIS
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- separates based on size and the smallest protein moves the fastest towards the positive pole
- one hour to run
- Run a standard mixture in one lane and samples in the others
- A dye is included so you can see how far the smallest component goes - ISOELECTRIC FOCUSING
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- an electrophoretic technique that separates proteins based on isoelectric point
- a stationary pH gradient is established
- Proteins are allowed to migrated in an electric field
- They migrate until they reach their isoelectric pH
- pH = pI - TWO DIMENSIONAL ELECTROPHORESIS
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- 1st dimension is isoelectric focusing so proteins are separated based on isoelectric pH
- 2nd dimension is SDS gel electrophoresis and separates everything by size
- SDS has to come after isoelectric because it turns every protein into negatively charged proteins - PROTEIN SEQUENCING
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- determine amino acid sequence present because proteins have a large number of amino acids
- determine the sequence of both ends first through FDNB
- Cleave protein into smaller pieces and determine some sequence using Edman Degradation
- purify fragments
- partial sequencing of fragments is performed
Combine all data on overlapping peptide sequences to complete the sequence - FDNB
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- FDNB reacts with any amino group to form a stable derivative that has a distinctive yellow color
- determines how many amino groups are present
- hydrolysis stage destroys polypeptide so no more sequencing can be done
- analyzes protein primary structure - EDMAN DEGRADATION
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- procedure labels and removes only the amino terminal residue from a peptide leaving all other peptide bonds intact
- cyclic molecule
- coupling step
- cleavage step
- extract ioin
- repeat as many times as necessary - SEQUENCING LARGE PROTEINS INTO SMALLER SEGMENTS
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- Break disulfide bonds
- Cleave the polypeptide Chain
- Sequence peptides
- Order peptide fragments
- locate disulfide bonds - MASS SPECTROMETRY
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- detects changges in mass of proteins due to the presence of bound cofactors, bound metal ions, covalent modified cations, and so on.
- A protein solution is dropped into a high voltage electic field
- droplets evaporate and ions enter the spectrometer
- Spectrum generates peaks corresponding to charged species - TANDEM MS
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- extracts sequence information
- protein solution is treated with protease or chemical reagent to hydrolyze it into shorter peptides
- Injected into a device
- peptide mixture is sorted and ionized fragments are manipulated so only one type of peptide emerges at the end.
- Peptide is fragmented with high energy impact with collision gas. Most breaks occur at peptide bonds
- Peaks are generated for peptide fragments - FMOC
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- synthesizes a peptide while keeping it attached at one end to a solid support
- the support is a insoluble polymer contained within a column
- at each step protective chemical groups block unwanted reactions
- protecting group is removed by flushing with mild organic base
- Alpha amino group of amino acid 1 attacks activated carboxyl group of amino acid 2 to form peptide bond
- Completed peptide is deprotected as in reaction 2
- HF cleaves ester linkage between resin