Midterm Prep

Question 1

Partition chromatography

Answer 1

▪ Particles of solid are chosen with a specific property, e.g. silica
gel has HO-Si-OH groups that can hydrogen-bond to polar
amino acids
▪ Stationary phase
Liquid solvent or buffer flows past the particles and is non-polar
Another format is thin layer chromatography

▪ Mobile phase

▪ Amino acids exchange (partition) between phases
▪ polar amino acids P spend more of their time hydrogen
bonded to silica and move slowly

Amino acids are identified by the volume of buffer needed
to move each through the column- elution volume

Question 2

Ninhydrin

Answer 2

Sprayed on thin layer chromotography plate to identify amino acids which show up purple

Question 3

Affinity Chromatography

Answer 3

• ligand (mlc with affinity for some amino acids) is in column
• proteins with afffinity for it bind and stay in column/bind tightly to it
• other proteins move faster and are eluted first
• high concentration of salt is added to displace and release the proteins bound to the ligand

Question 4

Affinity Chromatography

Answer 4

• ligand (mlc with affinity for some amino acids) is in column
• proteins with afffinity for it bind and stay in column/bind tightly to it
• other proteins move faster and are eluted first
• high concentration of salt is added to displace and release the proteins bound to the ligand

Question 5

Tag

Answer 5

A peptide or protein that binds a ligand with high affinity and specificity that is fused to the gene encoding target protein

Question 6

Amino acid analysis

Answer 6

Includes separation of a mixture into components and detection of components of interest
Can be qualitative and quantitative and preparative

Question 7

How are amino acids identified in partition chromatogra?

Answer 7

Illusion, volume concentration of amino acids is measured in each test cube, and the results are graphed

Question 8

Ion exchange chromatography

Answer 8

This is charged residence as a stationary phase cationic exchanger contains negative groups, which band deposit of molecules and ions and I am exchanger and he’s positive. Which binds to negative groups/anions

Question 9

How is illusion done in ion exchange chromatography

Answer 9

Hi ion concentration like a salt solution is inserted in common displace is the amino acid changing the pH to alter the charge on the iMac me know, I said, so no longer binds can also be done

Question 10

SDS polyacrylamide gel

Answer 10

Smaller proteins move faster than larger ones
It separates proteins that differ in molecular weight
An electric field is used to move the proteins through a gel
SDS denatures the proteins

Question 11

Urea

Answer 11

Weakens hydrophobic effect, allows protein to unfold/denature

Question 12

Fluorodinitrobenzene

Answer 12

Can be used to study protein sequence and puts a tag on the first (N-terminal) amino acid dying it yellow to be recognized
At high pH, this group deprotonates and hydrolysis releases it

Question 13

What factors cause certain amino acids to prefer beta

Answer 13

WYF VIT C
1) bulky amino acids prefer beta structure (WYF C)
2) awkward shaped/ branched amino acids also prefer it (VIT)

Question 14

The binding pocket of chumptrypsin is lined with nonpolar amino acids
What is this an example of?

Answer 14

Hydrophobic effect

Question 15

The binding pocket of chymotripsin is the right shape to fit a large amino acid
What is this an example of

Answer 15

This is an example of van der waals

Question 16

Isoelectric point

Answer 16

The pH at which the net charge of the protein is zero

Question 17

How many amino acids is each turn of the alpha helix

Answer 17

3.6

Question 18

How many ampheres is each turn of the helix

Answer 18

5.4

Question 19

Two dimensional gels allow the separation of

Answer 19

Proteins with similar isoelectric points that differ in molecular weight

Question 20

Importance of the alkaline phase in edman degradation

Answer 20

The N-terminal amino group becomes deprotonated under alkaline conditions making it nucleophilic and allowing it to react with the electrophilic PITC reagent

Question 21

Which catalytic mechanism requires the help of a cofactor or enzyme

Answer 21

Electrophilic catalysis

Question 22

Rate of enzyme reaction

Answer 22

Change in substrate concentration over time
= [S] / t
Units are umol/L/min

Question 23

Enzyme activity

Answer 23

Is the moles of substrate converted to product over time
= rate x volume
Units are umol/min

Question 24

Specific activity

Answer 24

= enzyme activity / mass of enzyme
Units are umoles/min/ug

Question 25

Turnover number

Answer 25

= specific activity x molar mass of the enzyme Units are s^-1

Question 26

Vmax

Answer 26

Is the limiting maximum rate as [S] tends to infinity Indicates the catalytic rate at which 100% of enzyme is occupied by substrate Vmax is not a true constant as it varies depending on the concentration of enzyme present Vmax = k2[E]tot

Question 27

Km

Answer 27

Indicates the affinity of the substrate for the enzyme Different Km for each substrate When v0= 1/2 vmax, the concentration [S] at this point gives the value of Km

Question 28

Low Km means

Answer 28

high affinity, the enzyme binds the substrate strongly

Question 29

High Km means

Answer 29

low affinity, the enzyme binds to the substrate weakly

Question 30

Higher Vmax means

Answer 30

faster reaction, better catalysis

Question 31

Fluorescamine

Answer 31

Similar to ninhydrin Gives yellow fluorescence under UV light

Question 32

Fluorescamine

Answer 32

Similar to ninhydrin Gives yellow fluorescence under UV light

Question 33

IMAC

Answer 33

Immobilized metal chromatography Clusters of His in a protein bind tightly to Ni2+ or Co2+ Column is made up of chelating resin containing Ni2+ His-tagged proteins bind tightly to Ni2+ resin The bound protein is eluted by adding imidazol (structure similar to His side chain) to the buffer High degree of purification in one step

Question 34

SDS Polyacrylamide gel

Answer 34

Used for electrophoresis 5-15% polymer and 90-95% water Separated proteins are visualized by adding a dye and then putting them in well All proteins have uniform net negative charge due to the sulphate of sds binding and separation is strictly based on size They travel from negative to positive Smaller goes faster/farther than larger

Question 35

Isoelectric focusing

Answer 35

Uses isolectric point to separate proteins Protein starts at high pH and negative top of electrode(gel with pH gradient) and as it moves down the gradient to lower and Lowe pH it stops at point where net charge is zero aka Isoelectric point

Question 36

Mass spectrometry

Answer 36

Provides a way to identify proteins Protein is vaporized by a laser beam yielding charged particles Particles travel toward the detector The time of flight to the detector yields very accurate mass measurement Mass of protein will be compared to database to identify

Question 37

Myoglobin

Answer 37

Protein with 8 alpha helices Four globin units for hemoglobin

Question 38

Proteases

Answer 38

Enzymes which catalyze hydrolysis of peptide bonds

Question 39

How May an atom use a lone pair

Answer 39

A hydrogen bond acceptor A base A nucleophile

Question 40

The Sanger method

Answer 40

To determine the amino acid sequence we can tag the Nterminus with fluorodinitrobenzene Big snag: hydrolysis destroys the rest of the polypeptide chain

Question 41

Edman degradation

Answer 41

N-terminal amino acid can be reacted, removed and identified without hydrolyzing the peptide bonds Involves two steps: 1. Coupling 2. Cyclization Reaction can be repeated ip to 50 times to give a 50 AA sequence reading

Question 42

Coupling step for edman degradation

Answer 42

Requires base Reaction must be complete before the next cyclization step can take place Deprotonated N terminal acts as a nucleophile with carbon from PITC, labelling it with that PITC)

Question 43

Cyclization stem edman degradation

Answer 43

Requires acid Cleaves the first peptide bond and it like makes a ring with the n terminus (Cyclization) to do this Now we can identify it Now we’re left with a shortened peptide (missing first AA) This reaction must be complete before the next coupling can take place

Question 44

New Km value equation for competitive inhibitors And new Vmax equation for non competitive inhibitor

Answer 44

Km’ = Km (1 + [I]/Ki) Vmax’ = Vmax / (1 + [I]/Ki)

Question 45

Slope of a line weaver beaker plot

Answer 45

= Km/Vmax Gets steeper when inhibitor present