Biochemistry Exam 2

Chapters 5-9

proteome

encompasses all functional proteins expressed in a cell, not a fixed characteristic, varies based on cell type and function

purification

the first step to understanding a proteins amino acid sequence, 3D structure, and function

assay

lab test performed to identify the presence of target protein, done after each step in purification process

cell lysis

the process of breaking open a cell, releases cellular contents and allows us to access our protein

homogenate

mixture of all components of a cell but no intact cells

differential centrifugation

lab process in which the homogenate of cell contents is centrifuged at a low speed to produce a pellet and supernatant; the supernatant is then centrifuged multiple times to yield several fractions of decreasing density, which still contain hundreds of proteins. They are assayed to check for the target protein. This process helps narrow down the amount of proteins present

salting in

dissolving proteins in salt

salting out

precipitating proteins out of a solution of high salt concentration

dialysis

process that removes high salt concentration from precipitated proteins, regaining activity

gel filtration

separates proteins by size. A protein mixture is added to column consisting of insoluble polymer beads; small molecules enter the beads, large molecules pass by through buffer and exit at the bottom first

ion exchange

separates proteins based on charge. If protein has net positive charge at pH 7, protein will bind to column of beads containing net negative charge and proteins with a negative charge will pass through the buffer

affinity chromatography

lab technique that takes advantage of proteins with a high binding affinity to specific chemical groups or molecules

high pressure liquid chromatography

process of improving column techniques’ ability to separate individual proteins. Beads are more finely divided, creating more interaction sites and a greater ability to separate.

gel electrophoresis

a lab technique used to separate, identify, and analyze DNA, RNA, and proteins based on size and charge. Applies an electrical field to the gel to pull charged molecules through. Small molecules will move faster and farther than larger molecules 

cathode

negative charge

anode

positive charge

which direction does the electrical charge in gel electrophoresis move

cathode to anode, + to -

SDS PAGE

used in gel electrophoresis, seperates proteins by size

dye commonly used to visualize proteins

coomassie blue

isoelectric point (PI)

point in which the pH of a protein is 0, at this pH the protein will not migrate through electric field

isoelectric focusing

method of separation where SDS PAGE is absent in gel electrophoresis, proteins will move until they reach the point where pH is equal to PI, when the protein has no net charge 

2D gel electrophoresis

powerful technique that separates proteins by isoelectric point and size

antibody

protein that is synthesized in response to presence of a foreign substance, called an antigen

polyclonal antibodies

mixture of antibodies produced by different B-cell clones in response to antigen

monoclonal antibodies

lab produced proteins that mimic the body’s natural antibodies, produced from 1 cell lineage by cloning a unique white blood cell

ELISA

immunological process of detecting and quantifying antibodies, antigens, or proteins. A specific antigen is immobilized on a plate, then the sample with the substance to be detected is added. If the target substance is present, it will bind to the antigen. a secondary enzyme is added to bind to the complex and a substrate is added to bind to that enzyme, causing color change

sandwich ELISA

highly specific lab technique used to detect and quantify specific proteins or antigens in a sample. Based on the principle of sandwiching the target protein or antigen between two antibodies, one immobilized on a plate and the other labeled with an enzyme. A substrate is then added, which binds to the antibody labeled with an enzyme, causing color change

What is the difference between ELISA and sandwich ELISA

ELISA detects the antibody, sandwich ELISA detects the antigen

western blotting

lab technique used to detect specific proteins in a complex mixture. Cells are lysed to extract proteins, then loaded on a gel to perform gel electrophoresis. After separation, the gel is transferred to a membrane, blocked with a solution to prevent antibodies from sticking non-specifically, and then the membrane is incubated with a primary antibody. A secondary antibody labeled with an enzyme is then added, which binds to primary antibody so it can be visualized

chemical cleavage

process of breaking chemical bonds between molecules such as proteins and nucleic acids using chemical reagents 

Enzymatic cleavage

highly specific biochemical process where enzymes break large molecules into smaller ones by breaking specific chemical bonds

What is the cleavage site of the chemical cleavage reagent cyanogen bromide?

carboxyl side of methionine residues

What is the cleavage site of the chemical cleavage reagent O-Iodosobenzoate?

carboxyl side of tryptophan residues

What is the cleavage site of the chemical cleavage reagent hydroxylamine?

asparagine-glycine bonds

What is the cleavage site of the chemical cleavage reagent 2-Nitro-5-thiocyanobenzoate

amino side of cysteine residues

What is the cleavage site of the enzymatic cleavage reagent trypsin?

carboxyl side of lysine and arginine

What is the cleavage site of the enzymatic cleavage reagent clostripain?

carboxyl side of arginine residues

What is the cleavage site of the enzymatic cleavage reagent staphylococcal protease?

carboxyl side of aspartate and glutamate residues

What is the cleavage site of the enzymatic cleavage reagent thrombin?

carboxyl side of arginine

What is the cleavage site of the enzymatic cleavage reagent chymotrypsin?

carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine

What is the cleavage site of the enzymatic cleavage reagent carboxypeptidase A?

Amino side of carboxyl terminal amino acid (not arginine, lysine, or proline) side

enzyme

a substance produced by a living organism, which acts as a catalysts for specific biochemical reactions

substrate

the molecule upon which an enzyme acts, the substrate binds to the enzyme’s active site to undergo specific chemical reaction

active site

a region on an enzyme that binds to a protein or other substance during a reaction

activation energy

the minimum amount of energy required for a chemical reaction to occur

rate limiting step

the slowest step in a multi step reaction or process that determines overall rate

equilibrium constant

a numeral value that expresses the ratio of products to reactants at equilibrium in a reversible chemical reaction

standard free energy change (ΔG°)

the change in gibbs free energy (ΔG) for a reaction where all reactants and products are in their standard states

binding energy 

the minimum energy required to seperate a system of particles into its individual components

induced fit

biochemical model where the active site of an enzyme changes its shape to bind a substrate more tightly

lock and key fit

biochemical model where the enzyme's active site has a fixed shape that is a perfect match for the shape of its substrate 

what are the differences between catalyzed and uncatalyzed reactions on an energy diagram

on an energy diagram a catalyzed reaction will have a lower action energy requirement than an uncatalyzed reaction

how can you tell the reactants are favored on an energy diagram

the potential energy of the reactants is lower than the products potential energy

how can you tell the product is favored on an energy diagram

if the final energy of the products is lower than the starting energy of the reactants

what is the difference between the lock-and-key and induced-fit models

the lock and key model assumes the enzyme’s active site has a rigid, predefined shape that perfectly matches the substrate, while the induced fit model proposes that the active site of an enzyme is flexible and changes it shape to better fit the substrate

enzyme substrate complex (ES)

a temporary molecular structure formed when an enzyme binds to its specific substrate at the active site

reaction velocity (V)

the rate at which a biochemical reaction converts reactants into products, measuring the speed of product formed over time

dissociation constant (Kd)

measures the strength of a biomolecular interaction by quantifying how strongly they bind. Represents the equilibrium concentration of a ligand needed to occupy hald of the binding sites on a receptor

maximum velocity (Vmax)

the theoretical maximum rate of an enzyme-catalyzed reaction, occuring when the enzyme is completely saturated with substrate

Michaelis constant (KM)

a measurement of an enzyme’s affinity for its substrate, substrate concentration when the reaction rate is half of the Vmax; a low KM means a higher affinity, and a high KM means a lower affinity, specific to each enzyme. KM 

What is the Michaelis-Menten equation?

V= (Vmax * S)/Km + S

what does the Michaelis-Menten equation describe

the rate of a single substrate enzyme-catalyzed reaction

competitive inhibition

type of enzyme inhibition where an inhibitor moelcule competes with the substrate for binding to the enzymes active site; increases Km, Vmax is unchanged

non-competitive inhibition

type of enzyme inhibition where an inhibitor molecule binds to a site on the enzyme other than the active site; Km unchanged

uncompetitive inhibition

occurs when an inhibitor binds inly to the enzyme-substrate complex, not the free enzyme, which prevents the conversion of substrate to product; decreases Km and Vmax

How will competitive inhibition look on an LB plot?

the slope of the line will change and the y-intercept will stay the same in the line with an inhibitor in comparison to the line with no inhibitor present

How will uncompetitive inhibition look on an LB plot?

the slope of the line will stay the same and the y-intercept will change in the line with an inhibitor in comparison to the line with no inhibitor present

how will non-competitive inhibition look on an LB plot?

the slope of the line and the y-intercept will both change in the line with an inhibitor in comparision to the line with no inhibitor present

What is a Lineweaver-Burk (LB) plot?

linear graph used in enzyme kinetics that plots 1/V versus 1/[S] to linearize the Michaelis-Menten equation. Helps determine the Vmax and michaelis constant

what are the similarities between myoglobin and hemoglobin

Myoglobin and hemoglobin are both oxygen-binding proteins, globular-shaped, containing an iron-containing heme group, and red in color. Both composed of polypeptide chains with 8 alpha helices

What is the difference between myoglobin and hemoglobin

Myoglobin is a monomeric protein (1 subunit) in muscle cells that stores oxygen, while hemoglobin is a tetrameric protein (4 subunits) in red blood cells that transports oxygen from the lungs to the tissues. Myoglobin has a higher affinity for oxygen than hemoglobin and myoglobin accepts oxygen from hemoglobin and releases it to mitochondria during exercise while hemoglobin picks up oxygen in the lungs are releases it to myoglobin in the muscle

how does oxygen binding change the structure of hemoglobin

The oxygen binds to one of the iron molecules in 1 of the 4 heme groups, which causes a structural shift from the tense state (T state) to the relaxed state (R state). The iron atom shifts from nonplanar to planar. This creates a more open conformation for higher affinity for oxygen 

fractional saturation equation

𝜃= [L]/Kd + [L]

Bohr effect

Acidic conditions cause the oxygen dissociation curve to shift right due to he hemoglobin having a lower affinity for oxygen in acidic conditions. Allows for more efficient oxygen delivery to tissues in need; seen in exercise when muscles produce lactic acid

P50 value in oxygen dissociation curve

The P50 value is the partial pressure of oxygen when hemoglobin is 50% saturated with oxygen; it is the measure of how readily hemoglobin binds and releases oxygen. Normal range in humans is between 25 and 29 mmHg; lower P50 higher affinity, higher P50 lower affinity

cooperative binding

the binding of a ligand to a protein with mutliple binding sites affects the affinity for subsequent ligands

sigmodial binding curve 

an S-shaped graph that shows oxygen saturation versus the partial pressure of oxygen; shape is the result of positive cooperative binding of oxygen to 4 heme site on hemoglobin 

what happens when a person is suffering from CO poisioning

CO binds to hemoglobin and blocks oxygen binding due to having a higher affinity than oxygen, this prevents oxygen from being transported throughout the body, starving it of oxygen and causing tissue hypoxia. CO also directly interferes with the mitochondria’s ability to use oxygen, preventing the production of ATP

Abbreviation of Alanine

Ala, A

Abbreviation of Arginine

Arg, R

Abbreviation of Asparagine

Asn, N

Abbreviation of Aspartic Acid

Asp, D

Abbreviation of Cysteine

Cys, C

Abbreviation of Glutamic Acid

Glu, E

Abbreviation of Glutamine

Gln, Q

Abbreviation of Glycine

Gly, G

Abbreviation of Histidine

His, H

Abbreviation of Isoleucine

Ile, I

Abbreviation of Leucine

Leu

Abbreviation of Methionine

Met, M

Abbreviation of Phenylalanine

Phe, F

Abbreviation of Proline

Pro, P

Abbreviation of Serine

Ser, S

Abbreviation of Threonine

Thr, T

Abbreviation of Tryptophan

Trp, W

Abbreviation of Tyrosine

Tyr, Y

Abbreviation of Valine

Val, V