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Biochem ❤️ > Midterm Prep > Flashcards

Midterm Prep Flashcards

1
Q

Partition chromatography

A

▪ 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

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2
Q

Ninhydrin

A

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

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3
Q

Affinity Chromatography

A
  • 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
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4
Q

Affinity Chromatography

A
  • 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
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5
Q

Tag

A

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

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6
Q

Amino acid analysis

A

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

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7
Q

How are amino acids identified in partition chromatogra?

A

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

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8
Q

Ion exchange chromatography

A

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

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9
Q

How is illusion done in ion exchange chromatography

A

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

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10
Q

SDS polyacrylamide gel

A

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

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11
Q

Urea

A

Weakens hydrophobic effect, allows protein to unfold/denature

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12
Q

Fluorodinitrobenzene

A

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

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13
Q

What factors cause certain amino acids to prefer beta

A

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

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14
Q

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

A

Hydrophobic effect

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15
Q

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

A

This is an example of van der waals

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16
Q

Isoelectric point

A

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

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17
Q

How many amino acids is each turn of the alpha helix

A

3.6

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18
Q

How many ampheres is each turn of the helix

A

5.4

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19
Q

Two dimensional gels allow the separation of

A

Proteins with similar isoelectric points that differ in molecular weight

20
Q

Importance of the alkaline phase in edman degradation

A

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

21
Q

Which catalytic mechanism requires the help of a cofactor or enzyme

A

Electrophilic catalysis

22
Q

Rate of enzyme reaction

A

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

23
Q

Enzyme activity

A

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

24
Q

Specific activity

A

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

25
Q

Turnover number

A

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

26
Q

Vmax

A

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

27
Q

Km

A

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

28
Q

Low Km means

A

high affinity, the enzyme binds the substrate strongly

29
Q

High Km means

A

low affinity,
the enzyme binds to the substrate weakly

30
Q

Higher Vmax means

A

faster reaction, better catalysis

31
Q

Fluorescamine

A

Similar to ninhydrin
Gives yellow fluorescence under UV light

32
Q

Fluorescamine

A

Similar to ninhydrin
Gives yellow fluorescence under UV light

33
Q

IMAC

A

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

34
Q

SDS Polyacrylamide gel

A

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

35
Q

Isoelectric focusing

A

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

36
Q

Mass spectrometry

A

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

37
Q

Myoglobin

A

Protein with 8 alpha helices
Four globin units for hemoglobin

38
Q

Proteases

A

Enzymes which catalyze hydrolysis of peptide bonds

39
Q

How May an atom use a lone pair

A

A hydrogen bond acceptor
A base
A nucleophile

40
Q

The Sanger method

A

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

41
Q

Edman degradation

A

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

42
Q

Coupling step for edman degradation

A

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)

43
Q

Cyclization stem edman degradation

A

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

44
Q

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

A

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

45
Q

Slope of a line weaver beaker plot

A

= Km/Vmax
Gets steeper when inhibitor present

Biochem ❤️ (4 decks)

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