Biochem Test 2

Breaking up tissue and releasing proteins by mechanical force (blender/homogenizer, mortar and pestle, sonicator, freeze/thaw etc.)

homogenization

Different speeds of centrifugation can isolate distinct organelles versus cytoplasm

differential centrifugation

For differential centrifugation a low speed of 600xg isolates…

nuclei and unbroken cells

For differential centrifugation a medium speed of 15000 xg isolates…

mitochondria and lysosomes

For differential centrifugation a high speed of 100,000 xg isolates…

ribosomes from ER, small vesicles

what is the step after differential centrifugation

Protein purification of supernatant/lysate

cytosolic/soluble proteins

lysate

High concentrations of salt cause water to prefer to interact with salt and become more ordered

salting out

>2M ammonium sulfate is considered ____ saturation

50%

when protein purification by ammounium sulfate happens ________ interactions are favored and then the protein becomes ________

hydrophobic, insoluble

what are the pros and cons of protein purification by ammonium sulfate

cheap and fast but purity can be lower

what are pros and cons of protein purification by column chromatography

more choices to yield pure protein, more expensive, more time

what are the two phases of column chromatography

stationary and mobile

• Immobile material of solid substance (called beads, resin, or matrix)

• Samples interact with this phase

stationary phase

Flows over the stationary phase and carries along with it the sample to be separated

mobile phase

• Uses specific binding properties of proteins

• Stationary phase has a polymer that can be covalently linked to a ligand that specifically binds to protein

affinity chromatography

what are the three steps of affinity chromatography

load (add proteins into mobile phase, ligand binds), wash (mobile phase removes unwanted proteins, protein stays bound to ligand), elute (add more mobile phase with ligand to elute protein from stationary phase)

• Protein separation based on charge.

• Stationary phase is modified to have different charges and intensity of charge at a defined pH – positive or negative.

ion exchange chromatography

negatively charged resin; exchange of cations (isolate positively charged proteins)

cation exchange chromoatography

positively charged resin; exchange of anions (isolate negatively charged proteins)

anion exchange chromatography

• Also called gel filtration

• Protein separation based on size/molecular weight.

• Stationary phase composed of cross-linked gel particles where the extent of cross- linking determines the pore size and density

• Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter the porous beads and elute from column first.

size exclusion chromatography

• Medium pressure instrument with computerized pumps and detectors

• Monitor mobile phase by UV absorbance to determine when protein elutes from column. Collect specific volumes in tubes/fractions)

fast protein liquid chromatography

• Powerful technique to determine purity of protein sample

• Separates proteins based on size, charge, and shape/conformation

• Technique involves loading sample into a gel matrix (typically polyacrylamide for proteins) and applying an electric field

• charged particles migrate in electric field toward opposite charge

gel electrophoresis

• Separation is based on size

• Add negatively charged SDS denatures protein structure and imparts a uniform negative charge on proteins

• Add Beta-mercaptoethanol – break up disulfide bonds

• type of gel electrophoresis

SDS PAGE

• No SDS/No BME, proteins separated by size and charge

• Retains quaternary structure of protein but sometimes more complicated due to charge differences

Native PAGE

how do you assess purity

run standards and visualize bands

dye that stains gel with preference for tryptophan, argininge, and phenalaline

coomassie blue

• Separation based on differing isoelectric points (pI) of proteins

• Gel is prepared using ampholytes (small molecules that can exist in different charge states depending on pH). Creates a stable pH gradient when electric field is applied.

• Charge on the protein changes as it migrates through the pH gradient.

• When it gets to pH = pI, has no charge and stops migrating

isoelectric focusing

• Type of protein made by immune system as your body’s natural defense system

• Neutralize pathogens by binding to the pathogens or “antigens” and preventing activity

antibodies

the most common antibody has a ___ like structure

Y

Type of enzyme/antibody assay used to detect protein amounts as low as picograms

ELISA

what are the steps for an elisa

imobilize protein, add primary antibody, add secondary antibody (used for visualization), add substrate specifc for enzyme (color change)

What tells you the amount of protein/antigen in an elisa

how much color change

Another type of enzyme/antibody assay used to detect protein amounts as low as picograms uses membrane (special sheet of paper) instead of sample well format

western blot

What are the steps of a western blot

run electrophoresis, transfer proteins to memebrane, add primary antibody, add secondary antibody which is conjugated to enzyme, add substrate and detect signal

• Determines the amino acid sequence or primary structure of a protein

• Can also be used to determine amino acid modifications, like phosphorylation

amino acid sequencing

what are some cons of amino acid sequencing compared to dna sequencing

more challenging and time consuming with overlapping steps

What is step one of amino acid sequencing

hydrolyze proteins into individual amino acids (Use HCl and heat to cleave peptide bonds, use hplc to see number and type of amino acids)

reaction in presence of water that results in cleavage of bonds

hydrolosis

What is step 2 of amino acid sequencing

edman degredation (n terminal protein sequencing on single polypeptide chain)

what chemicals are used in edman degredation and what happens after

Phenylisothiocyanate, triflouroacetic acid, weak aqueous acid (yields n terminus as pth derivative that can be identified by hplc)

what is step 3 of amino acid sequencing

cleavage at specific bonds

Cleavage by enzymes: Trypsin, Chymotrypsin, Elastase, thrombin, pepsin

peptidase cleavage

Cleavage by chemicals: Cyanogen bromide

chemical cleavage

Cleaves at C-terminal of positively charged side chains (Arginine and Lysine)

trypsin

Cleaves at C-terminal of aromatics (Tyr, Trp, Phe)

Chymotrypsin

cleaves at C-terminal of internal Methionine to make homoserine lactone at C-terminal and reducing the overall peptide length

cyanogen bromide

• a heterogeneous class of naturally occurring organic compounds classified together based on solubility

• Insoluble in water

• Soluble in aprotic organic solvents including diethyl ether, chloroform, methylene chloride, and acetone

• Amphipathic in nature (one polar/one nonpolar)

lipid

what are the open chain forms of lipids

fatty acids, triacylglycerols, phosphoacylglycerols, sphingolipids, waxes, eicosanoids

what are the cyclic chained forms of lipids

cholesterol, steroid hormones, and bile acids

what are the mixed chained forms of lipids

vitamin E and K

• an unbranched hydrocarbon- chain with a carboxylic acid head

• most commonly consists of an even number of carbons, approximately 12 - 20 carbons in length

fatty acid

fatty acid with no double bonds between carbons in the chain

saturated fatty acids

fatty acid with one double bond between carbons in the chain

monosaturated fatty acid

fatty acid with more than one double bond in the chain

polyunsaturated fatty acid

Saturated fatty acids (no double bonds) tend to adopt more ______ conformations

linear

Unsaturated fatty acids (double bonds) tend to adopt more ____ conformations

bent

what are triglycerols made from

3 fatty acids, glycerol

what are glyerocophopholipids made from

2 fatty acids, glycerol, phospholipid, alcohol

what are spingolipids (phospholipid) made from

spingosine, fatty acids, phosphate

what are spingolipids (glycolipid) made from

spingosine, fatty acid, sugar

What factors make a fatty acids melting point increase

longer chain and saturated

a lower melting point in a lipid means it is more…

liquid like

a long chain will make water solubility…

decrease

_______ are the most abundant lipid

triacylglycerols

what are solid triacylglycerols

fats

what are liquid triacylglycerols

oils

Triacylglycerols are less soluble in water than fatty acids due to the lack of …

charged carboxylate group

what is the second most abundant lipid

Glycerophospholipids

what are the polar alcohol head groups in Glycerophospholipids

choline, ethanolamine, serine, glycerol, inositol

an 18- carbon amino alcohol with both an amino group (–NH₂) and a hydroxyl group (–OH)

sphingosine backbone

the simplest sphingolipid is a cermaide consisting of…

sphingosine backbone and a fatty acid

where are sphingolipids commonly found

cell membrane

the most common sphingolipid is a…

sphingophospholipid/ sphingomylein

what does a sphingomylein consist of

Phosphocholine polar head group, Sphingosine backbone, Fatty Acid

sphingomylein are the primary component of the _________ in nerve cells

mylein sheath

• a compound in which a carbohydrate (sugar) is bound to an -OH of the lipid

• Sugar is often glucose or galactose

glycolipid

• sphingoglycolipids

• one sugar as the head group

cerobrosides

• sphingoglycolipids

• two sugars as the head group

globosides

• sphingoglycolipids

• more complex oligosacharride head group

• least one sialic acid residue (N-acetylneuraminidate acid)

gangliosides

which Sphingoglycolipids is an important component of the neuronal cell membrane

Ganglioside

Buildup of glucocerebroside

Gaucher disease

Buildup of sphingomyelin

Niemann-Pick disease

Buildup of gangliosides

Tay-Sachs disease

• Fatty acid + Long chain Alcohol (Ester linked)

• Non-polar, found as protective coating for plants and animals

• High melting points à Typically solid at room temperature

waxes

• Naturally-occurring substances derived from 20- carbon polyunsaturated fatty acids

• Involved in hormone signaling

eicosanoids

• 5-carbon ring

• pain/inflammation

• eicosanoids

Prostaglandins

• 6-membered ring with ether

• blood flow and clotting

• eicosanoids

thromboxanes

• 3 conjugated double bonds

• Expressed in white blood cells (leukocytes)

• eicosanoids

Leukotrienes

• lipid comprised of 2 or more isoprenes

• 5 carbon molecule

Isoprenoids

what are the three types of isoprenoids

Terpenes, Steroids, Lipid Vitamins

• 17-carbon tetracyclic ring (3x six-membered ring + five- member ring)

• Biosynthetically derived from isoprene units

• Sterol – hydroxyl at the C3 position

steroid

whta is the most abundant teroid and what is it important for

cholesterol and membrane rigidity

Essential nutrients that humans can’t synthesize

lipid vitamins

what are the lipid vitamins

A E K D

animal membranes contain _______ which is not found in plant membranes

cholesterol

how is cholesterol a key component in membrane fluidiity

high temperatures prevent phospholipids from becoming liquid, in low temperatures prevent phospholipids from packing to tightly

describe the fluid mosaic model

fluid lateral motion of components, mosic components exist side by side

• Do not span or embed the membrane

• Bound by electrostatic interactions, hydrogen bonding, or anchored to the outside lipid molecule

• Removed by raising the ionic strength + detergents

Peripheral membrane proteins

• Span or embed within the membrane

• Bound tightly to the interior of the membrane

• Removed using detergents (denatured)

integral membrane protein