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Biochem > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

purification

A

isolate 1 protein from all others
-generally use freshly isolated cells or tissues
-exploit solubility, size, charge and/or specific binding affinity for protein of interest

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2
Q

cell fractionation

A

breaking cell into pieces
-remove contaminating proteins, until protein activity as close to 100% as possible
-increasing specific activity

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3
Q

protein purification steps

A

choose source, obtain large supply
lyse cells, centrifuge to obtain functions
salting out
dialysis
chromatography
analyze purity and activity

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4
Q

lysis

A

disruption of cells

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5
Q

centrifugation

A

increase concentration and purity target protein in sample
-spin at high force to separate
-large and more dense settle to bottom

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6
Q

salting out

A

add increase concentration of salt
-exploit differing solubility between proteins
-more ions= more polar solution becomes

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7
Q

dialysis

A

removes salt to resolubilize protein
-also removes small contaminating molecules
-pick optimal pore size, buffer makeup and pH

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8
Q

gel filtration chromatography

A

size exclusion chromatography
separates proteins based on size and shape (least selective)
large proteins migrate faster than small, could get trapped in carb beads

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9
Q

ion exchange chromatography

A

exploits charge differences
typically elute proteins with counter ion, gets proteins to stick

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10
Q

anion ion chromatography

A

binds neg charged proteins to + charged matrix

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11
Q

cation ion chromatography

A

binds pos charged proteins to - charged matrix

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12
Q

affinity chromatography

A

exploits specific binding properties of target protein
elute protein with free ligand
most specific way to separate proteins

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13
Q

solid stationary phase

A

moves slowly

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14
Q

liquid mobile phase

A

moves more quickly

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15
Q

fraction

A

what collected during chromatography

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16
Q

His-tag

A

bunch histidine’s in a row
resin (matrix) with nickel added NTA groups attached
use imidazole to remove

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17
Q

native PAGE

A

retains biologically active shape
separation by charge, size, shape

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18
Q

SDS PAGE

A

separates by mw
unravels protein and gives - charge

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19
Q

reducing

A

contain added reducing agents to destroy disulfide bonds

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20
Q

non-reducing

A

no added reducing agents- disulfide bonds remain intact

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21
Q

percentage in PAGE

A

tells us % crosslinking occurred and helps determine what to use depending on size

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22
Q

1D

A

separate top to bottom
use to determine how many aa

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23
Q

2D

A

pI focus gel with pH gradient

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24
Q

edman degradation steps

A

label protein N terminal amino group and cleave between 1st and 2nd amino acid
can sequence up to 50 amino acid residues

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25
Q

amino acid hydrolysis

A

amino acid composition and ratios

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26
Q

overlap fragments

A

1st find common start
2nd find common stop
3rd use overlap

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27
Q

mass spectrometry

A

measure mass to charge ratio
-measures mass of small peptides

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28
Q

ESI-MS

A

electrospray ionization
-based on mass and acceleration through chamber ending in a detector
-the sample becomes ionized due to high voltage
-ionization evaporates the solvent, leaving peptide in the gas phase to travel through separating mass spectrometer
-high voltage can be used to fragment the sample
-uses mass to charge ratio to determine molecular mass
-can be used for proteins that have been treated with proteases like trypsin

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29
Q

MALDI-MS

A

matrix-assisted laser desorption/ionization
-based on mass and acceleration through chamber ending in a detector
-peptides become fragmented and charged due to laser exposure
-can be used for proteins that have been treated with proteases like trypsin
-uses mass to charge ratio to determine molecular mass
-proteins or peptides are mixed in a solid matrix rather than a solution
-laser will be absorbed by the peptide containing material releasing the peptide into the gas phase

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30
Q

Tandem MS

A

used to identify aa sequences
ms twice

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31
Q

collision chamber in ms/ms

A

divides peptide fragment into smaller subunits

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32
Q

function of second ms

A

determine the masses of peptide subfragments

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33
Q

function of first ms

A

determine the masses of the protease digestion fragments

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34
Q

xray crystallography

A

high resolution structures
static
has no limit
requires large sample concentration
high artifacts

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35
Q

NMR

A

low resolution
dynamic
low risk of artifacts
size limiting
allows for real-time ligand binding
requires large concentration of sample

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36
Q

Cryo-EM

A

high resolution
static
no size limit
requires small concentration of sample
can determine structure of multiple conformations

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37
Q

antibodies

A

recognize protein as antigen

38
Q

epitope

A

specific site on antigen that can bind to antibody

39
Q

Polyclonal

A

heterozygous mixture of antibodies
-recognize multiple epitopes of an antigenic protein
-typically more affordable, limited supply
-produced in rabbits, antibodies harvested from blood

40
Q

monoclonal

A

homozygous antibody
-recognizes only one epitope of antigenic protein
-more expensive, unlimited supply
-typically produced in mice, antibodies harvested from hybridomas

41
Q

western blotting steps

A

separate proteins by SDS-PAGE
transfer proteins to thin membrane
block nonspecific binding using milk
incubate with primary antibody
incubate with secondary antibody-enzyme conjunction
detect antigen-antibody complex

42
Q

western blotting

A

used to detect protein
determines if protien is there

43
Q

immunofluorescence

A

used to determine where protein is in a cell

44
Q

ELISA

A

used to quanify an antigen or antibody in a sample

45
Q

indirect ELSIA

A

similar to western blot concept
used to detect either antigen or primary Ab

46
Q

sandwich ELISA

A

similar to indirect, except antigen is captured by antibody instead of being directly bound to the surface

47
Q

immunoprecipitation

A

uses SDS-PAGE and MS to identify unknown proteins

48
Q

metabolic enzymes

A

reaction catalyst that control metabolic flux
-lower activation energy
-higher reaction rate
-do not alter equilibrium concentration of products and reactants

49
Q

structural proteins

A

maintain structural integrity and promote changes in shape
-framework for cells, tissues, and organs
*actin
*tubulin

50
Q

cell signaling proteins

A

transmit extracellular and intracellular signals by functioning as switches

51
Q

genomic caretakers

A

maintain integrity and accessibility of genomic info
-includes proteins involved in DNA replication, repair and recombination (DNA poly, DNA ligase etc)
-involved in gene expression

52
Q

transport proteins

A

move molecules within and between cells

53
Q

Myoglobin (mb)

A

concentrated in muscle, storage depot for O2
-monomer w one heme

54
Q

Hemoglobin (hb)

A

make up RBC
transport O2 from lungs to tissues with circulatory system
-tetramer w 4 heme (2 alpha, 2 beta)

55
Q

tense state

A

larger opening in the center

56
Q

ligand binding

A

reversible process involving noncovalent interaction
induces or stabilizes structural conformation in target protein

57
Q

O2 binding curve for Hb

A

sigmoid which indicates cooperative binding
(+ modulation)

58
Q

allosterism

A

when binding of ligand at one site effects conformational change at another site of same protein

59
Q

modulators

A

ligands that induce allosterism changes
can be + or -

60
Q

homotropic modulator

A

identical ligand and modulator

61
Q

heterotrophic modulator

A

ligand different from modulator

62
Q

CO binding to Hb

A

stronger affinity for CO causes less O2 binding
supports R state
-very high affinity for O2 but won’t let go, lose sigmoid

63
Q

Ph and CO2 binding to Hb

A

Protons bind to several residues-helps stabilize tense state and adjusts salt bridges
-CO2 added to N terminal amino group of each chain
-decrease affinity of Hb for O2

64
Q

2,3 BPG binding to Hb

A

one molecule per Hb tetramer, binds to central cavity
reduces affinity for O2
stabilizes T state

65
Q

maternal Hb

A

2 alpha 2 beta
typical 2,3 BPG binding

66
Q

fetal Hb

A

2 gamma 2 alpha
reduced affinity for 2,3 BPG, increases oxygen affinity

67
Q

anemia

A

reduced oxygen transport efficiency from the lungs to the tissue
-caused by altered Hb function or reduced number of RBC
-can also be due to mutation in alpha or beta subunits

68
Q

sickle cell anemia

A

recessive genetic disease
valine for glutamic acid substitution at position 6 in beta globulin
heterozygous for trait more resistant to Malaria

69
Q

passive transport

A

facilitate movement down concentration gradient
-does not require energy input
(-)delta G

70
Q

active transport

A

move biomolecules against concentration gradient
-requires additional energy to induce conformational change in protein to open or close gated channel

71
Q

facilitated diffusion

A

moves down concentration gradient

72
Q

simple diffusion

A

nonpolar crosses membrane on own

73
Q

ionophore

A

pick up ion, shield charge and move into cell, release

74
Q

ion channel

A

open and let move down concenration gradient

75
Q

primary active transport

A

driven by ATP against concentration gradient

76
Q

secondary active transport

A

one downhill to drive other uphill

77
Q

carrier

A

saturable and transport at rates much slower than diffusion

78
Q

K+ channel

A

passive transporter
channel, non saturable
K+ desolvated by carbonyl positioning, energy source is movement of K+ down gradient (linear graph)

79
Q

glucose transporter

A

passive transporter
carrier, saturable (only move one at a time)
energy source is glucose moving down gradient (plateaued graph)

80
Q

P-type transporter

A

use phosphorylation to drive protein conformational changes
-carrier, saturable, ATP-driven transport

81
Q

PMCA

A

move Ca2+ using ATP

82
Q

SERCA

A

in muscle or cells, move Ca2+ using ATP

83
Q

N domain

A

nucleotide domain
where ATP (nucleotide) docks

84
Q

p domain

A

phosphoryl domain
one phosphorylated during mechanism
Accepts the phosphate from ATP during the catalytic mechanism

85
Q

a domain

A

acutator domain
translates conformational changes to M domain
Communicates the movement between the P and M domains

86
Q

m domain

A

membrane domain
where Ca2+ crosses membrane
Contains the substrate binding sites, allowing for transport from one side of the membrane to the other

87
Q

Na+ I- symporter

A

primary active transports generates the Na gradient, example of secondary active
helps import I- into cell to produce thyroid hormone
saturable, pleatued graph

88
Q

symport

A

same direction

89
Q

antiport

A

opposite directions

90
Q

muscle contraction steps

A

Calcium is released from the sarcoplasmic reticulum.
Calcium binds to troponin, uncovering myosin binding sites on the actin thin filaments.
Inorganic phosphate is released, triggering the power stroke.
ADP is released from the myosin head group
ATP docks onto the myosin head group.
The myosin head group is triggered to dissociate from actin.
ATP hydrolysis induces the recovery conformation.

91
Q

secondary active transporter

A

used to co transport second molecule uphill
energy source: movement of molecule down its electrochemical gradient

Biochem (10 decks)

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