Principles of Biochemistry Exam 2

Homogenization

the process of breaking cells open to release organelles

Types of homogenization

• blender + buffer = breaks some organelles

• Potter-Elvehjen homogenizer (tube + tight fitting plunger) = breaks open cell while leaving organelles intact

• Sonification = sound waves

• freezing/thawing 

Percent recovery

measurement of the amount of enzyme recovered at each step of purification

    = Total activity/Initial total activity

Specific activity

= Total activity/Total protein

Differential Centrifugation

Used to separate cell components into supernatant and pellet of organelles

Steps of Differential Centrifugation

1) spun at 600g x 10 mins to release nuclei and unbroken cells

2) spun again at 15,000 g x 5 mins to release mitochondria, lysosomes, and microbodies

3) spun again at 100,000 g x 60 mins to release ribosomes, endoplasmic reticulum, Golgi body, plasma membrane fragments in pellet and soluble cytoplasm in supernatant. 

Salting out

purification technique for proteins based on solubility in salt (ammonium sulfate) solutions. 

Salting out procedure

Salt removes water from proteins, resulting in hydrophobic interactions among the proteins

Salt is added in increasing saturation increments (40%, 60%, 70%) and spun to release protein of interest

As salt concentration increases, solubility decreases

Size-Exclusion/Gel-Filtration

used to separate molecules based on size via cross-linked gel particles/beads, which can be controlled to determine pore size.

Steps of Size-Exclusion/Gel-Filtration

1) mixture of molecules of varying size added to filtration column

2) large molecules flow through quickly while small molecules get stuck in cross-linked gel particles/bead pores

Affinity Chromatography

filters proteins based on specific binding properties using ligands

Steps of Affinity Chromatography

1) sample with mixture of proteins added to filtration column

2) protein of interest binds to ligand

3) other proteins filter out

4) protein of interest + ligand elute from column

Ion-Exchange Chromatography

used to filter proteins by overall protein charge (less specific than affinity). 

Cation exchange (neg charged column binds to pos charged proteins)

Anion exchange (pos charged column binds to neg charged proteins)

Steps of Ion-Exchange Chromatography (Ex: cation)

1) column resin (neg) is bound to Na+

2) proteins with 0 or neg net charge pass through column while pos stick to column, displacing Na+

3) excess Na+ added to outcompete the pos proteins which then elute

Gel electrophoresis

charged particles migrate in electric field toward opposite charge based on protein charge, size, and shape.

matrix for nucleic acids: agarose

matrix for proteins: polyacrylamide (more resistance toward larger molecules over small)

proteins are treated with sodium dodecyl sulfate as detergent (SDS)

Two-dimensional gel electrophoresis

protein bands separated by charge THEN size using isoelectric focusing and SDS-PAGR

Isoelectric focusing

separation by charge on the basis of isoelectric points (pH at which molecules have zero net charge). Used in 2D gel electrophoresis

SDS-PAGE

(SDS-polyacrylamide gel electrophoresis) used to separate by size in 2D gel electrophoresis 

How to determine primary structure

1) Separate and identify individual amino acids
2) Determine the N- and C- termini of the sequence (a.a sequencing)
3) Determine the sequence of smaller peptide fragments

HPLC – High Performance Liquid Chromatography

chromatography technique that gives fast and clean purifications

Trypsin

enzyme that cleaves peptide bonds for lysine and arginine so that the C- terminal amino acid ends up pos charged

Chymotrypsin

enzyme that cleaves peptide bonds for tyrosine, tryptophan, and phenylalanine so that the C-terminal amino acid ends up aromantic

Cyanogen Bromide

enzyme that cleaves peptide bonds for methionine so that the sulfur of the methionine and the carbon of the enzyme react to produce a homoserine lactone at the C-terminal

 Steps of peptide sequencing by the Edman method

1) Phenylisothiocyanate (Edman’s reagent) + mildly alkaline conditions + the N-terminus = phenylthiocarbamoyl substitution

2) phenylthiocarbamoyl substitution + trifluoroacetic acid (TFA) = resleases N-terminal AA as a thiazolinone derivative

3) Thiazolinone derivative + aqueous acid = N-terminal AA as a PTH
derivative (which can be read by HPLC)

4) Repeat with each “new” N-terminal until entire peptide is sequenced

Enzyme

A biological catalyst. Most usually a globular protein, though sometimes RNA. 

Increases rate of reaction up to 10 ²⁰ vs. uncatalyzed

Lowers activation energy

Some are specific to only one stereoisomer, others can catalyze many similar reactions

How does temperature effect reactions?

Increases rate of reaction. Eventually leads to protein denaturation.

Enzyme-Substrate Complex

Substrate: a reactant

Active site: portion of enzyme surface where the substrate(s) becomes bound by noncovalent forces (hydrogen bonding, electrostatic attractions, van der Waals attractions)

Lock-and-key model

substrate binds to that portion of the enzyme with a complementary shape

Induced fit model

binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

Chymotrypsin

catalyzes selective hydrolysis of peptide bonds where the
carboxyl is contributed by Phe and Tyr as well as hydrolysis of the ester bonds

Non-Allosteric Enzyme Behavior

Point at which the rate of reaction does not change, enzyme is saturated, maximum rate of reaction is reached

Michaelis-Menten Kinetics Equation

used to account for kinetic properties of enzymes

V = V_max[S] / K_M + [S]

where K_M is the dissociation constant for ES (greater the value of K_M the less tightly S is bound to E) and V_max is the theoretical maximum velocity (never actually achieved)

First order

a reaction whose rate depends on the power of the concentration of the reactant

Zero-order

a reaction whose rate does not depend on the power of the concentration of the reactant

Steady State

when rate of enzyme-substrate complex is equal to the rate of it’s breakdown

Michaelis-Menten Kinetics Equation Linearly

reciprocal of hyperbola equation

1/v₀ = (K_M / V_max [S]) + (1/V_max)

AKA Lineweaver-Burk double reciprocal plot

Turnover number

k_cat = (V_max / [E_T]) where E_T is enzyme concentration when all enzyme molecules are binding the substrate

Lineweaver-Burk Plot

X-axis: 1/S

Y-axis: 1/V

slope of line = K_M/ V_max

X-intercept = -1/K_M

Y-intercept = 1/V_max

Reversible inhibitor

Substance that binds to an enzyme to inhibit it, but can be released

Competitive inhibitor

Binds to the active (catalytic) site and blocks access to it by substrate. Substrate must compete with inhibitor, so more substrate is required. Results in K_M change on Lineweaver-Burk plot.

Noncompetitive inhibitor

Binds to a site other than the active site; inhibits the enzyme by changing its conformation. Results in V_max change while K_M remains the same

V^I_max = V_max / 1 + [I]/K_f

Irreversible inhibitor

A substance that causes inhibition that cannot be reversed. Usually involves formation or breaking of covalent bonds to or on the enzyme

Uncompetitive inhibitor

can bind ONLY to the ES complex but not to free E. V_max and K_M decrease.

Allosteric enzyme

an oligomer whose biological activity is affected by other substances binding to it, altering its 4°structure. Results in a sigmoidal curve in a plot of reaction velocity vs. substrate concentration.

Allosteric effector

a substance that modifies the behavior of an allosteric
enzyme; may be an allosteric inhibitor or allosteric activator

Feedback Inhibition

final product of chain reaction blocks and early reaction and shuts down whole series

Organization of Aspartate transcarbamoylase (ATCase)

catalytic unit: 6 subunits

organized into 2 trimers

regulatory unit: 6 subunits

organized into 3 dimers

Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cysteine (p- hydroxymercuribenzoate)

K system

an enzyme for which an inhibitor or activator alters K_0.5

V system

an enzyme for which an inhibitor or activator alters V_max but not K_0.5

Homotropic effects

allosteric interactions that occur when several identical molecules are bound to the protein (Ex: the binding of aspartate to ATCase)

Heterotropic effects

allosteric interactions that occur when different substances are bound to the protein (Ex: inhibition of ATCase by CTP and activation by
ATP)

Concerted Model

In changing from T to R and vice versa, all subunits change conformation simultaneously

C = KR/KT
KR indicates the affinity of R to S
KT indicates the affinity of T to S
C = Ratio of dissociation constants

Higher c = higher affinity between S and R form = less sigmoidal.

L = ratio of T and R form

Higher L = favorability of free T form = more sigmoidal.

T (tight or taut) conformation

binds substrate less tightly; the inactive form (absence of substrate) stabilized by allosteric inhibitor (I)

R (relaxed) conformation

binds substrate tightly; the active form (shifts in presence of substrate) stabilized by allosteric activator (A)

Sequential Model

the binding of substrate induces a conformational change from the T to the R form as per the induced-fit model of substrate binding

Zymogen

Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme

Chymotrypsinogen

synthesized and stored in the pancreas, which when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give pi-chymotrypsin

Coenzyme

a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction. (metal ions or vitamins)

Lipids

heterogeneous class of naturally occurring organic compounds classified on the basis of solubility. (Insoluble in water, but soluble in organic solvents including diethyl ether, chloroform, methylene chloride, and acetone)

Open Chain lipid forms

fatty acids, triacylglycerols, sphingolipids, phosphoacylglycerols, glycolipids, lipid-soluble vitamins, prostaglandins, leukotrienes, and thromboxanes

Cyclic lipid forms

cholesterol, steroid hormones, and bile acids

Unsaturated Fatty Acids

unbranched-chain carboxylic acid that contain C=C bonds. Lower melting point than saturated (greater the degree of unsaturation, lower the melting point)

Cis > Trans configuration

plant membranes have a higher percentage than animal membranes

(notated as: # of carbons: # of double bonds in the chain).

Saturated Fatty acids

unbranched-chain carboxylic acid that contain C—C bonds. Higher melting point than unsaturated.

(notated as: # of carbons: # of double bonds in the chain).

Triacylglycerol

an ester of glycerol with three fatty acids

Phosphoacyclglycerols

Phosphatic acid with another alcohol esterfied to the phosphoric acid. Found in plants and animals.

Steroids

group of lipids that have fused-ring structure of 3 six-membered rings, and 1 five-membered ring

Phosphoglyceride lipid bilayer

polar hydrophilic head groups are in contact with the aqueous environment, while nonpolar hydrophobic tails are within the bilayer

hydrocarbon tails in the interior can be rigid (if rich in saturated fatty acids) or fluid (if rich in unsaturated fatty acids

Biological membranes

• with heat, membranes become more disordered; the

transition temperature is higher for more rigid membranes;

it is lower for less rigid membranes

• the membranes of prokaryotes, which contain no

appreciable amounts of steroids, are the most fluid

Effect of cholesterol on lipid bilayer

reduces fluidity by stabilizing extended chain conformations of the hydrocarbon tails of fatty acid. is characteristic of animal rather than plant membranes

Peripheral membrane proteins

bound by electrostatic interactions. Can be removed by raising the ionic strength.

Integral proteins

bound tightly to the interior of the membrane. Can be removed by treatment with detergents or ultrasonification, denaturing them.

Fluid Mosaic Model

Components in the membrane have lateral motion and exist side-by-in as separate entities

Model