1
Columbia Biological Society
First meeting:Tuesday Sep 25
at 9pm in 702 Hamilton.
Note: Graphic 24 has been modified from the original posting:
(“Most spherical” was replaced by “least spherical”).
2
Riboflavin~ vitamin B2
Heme
Pyridoxal phosphate~ vitamin B6
Tetrahydrofolic acid~ vitamin B9
Some prosthetic groupsParticular small molecules so tightly bound that they are always found associated with the protein
3Membrane proteins
Could be size selective
Could be size and charge selective
Anion: an ion that would migrate to the anode in an electric field
4Small molecules bind with great specificity to pockets on protein surfaces
Too far
5Estrogen receptor binding estrogen, a steroid hormone
estrogen estrogen
detail
6Protein separation methods
Ultracentrifugation
Mixture of proteins
7centrifugal force = m(omega)2r
m = massomega = angular velocity r = distance from the center of rotation
Causing sedimentation:
Opposing sedimentation = friction = foV.
Constant velocity is soon reached: centrifugal force = frictional force So: m(omega)2r = foV
fo = frictional coefficient (depends on shape)
And: V = m(omega)2r/fo,
Or: V = [(omega)2r] x [m / fo] V proportional to mass (MW)V inversely proportional to fo (shape)V inversely proportional to non-sphericity(Spherical shape moves fastest)
Note: formulas wil be provided on exams, as will formulae
8Ultracentrifuge
9
Large, +++high positive charge
Large, +low positive charge
Small, +++High positive charge
Small, +Low positive charge
Molecules shown after several hours of electrophoresis
Glass plates
+
+ +
+++ ++
+
+++
+
+
+
+
++
+ ++
+ +++ +
+
10
Molecules shown after several hours of electrophoresis
Glass plates
Molecules shown after several hours of electrophoresis
Glass plates
+
+ +
+++ ++
+
+++
+
+
+
+
++
+ ++
+ +++ +
+
Winner: Small, +++High positive chargeLoser:Large, +low positive chargeIntermediate:Large, +++high positive chargeIntermediate:Small, +Low positive charge
11
12
Reservoir for bufferElectrode connection
Clamped glass sandwich
13
Tracking dyes
Happy post-doc
Power supply
Electrodes
14
15SDS PAGE = SDS polyacrylamide gel electrophoresis
• sodium dodecyl sulfate, SDS (or SLS): CH3-(CH2)11- SO4-
• CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SO4-
SDS
All the polypeptides are denatured and behave as random coilsAll the polypeptides have the same charge per unit lengthAll are subject to the same electromotive force in the electric fieldSeparation based on the sieving effect of the polyacrylamide gelSeparation is by molecular weight onlySDS does not break covalent bonds (i.e., disulfides)
16Summary of SDS PAGE
Separates on MW only, no shape no charge
High resolution.
Can measure the MW of a protein (subunit MW) by comparig mobiltiy to that of standards.
Must first reduce any disulfides to get true subunit MW (e.g., with mercaptoethanol).
17
Sephadex bead
Molecular sieve chromatography(=gel filtration, =Sephadex chromatography)
18
Sephadex bead
Molecular sieve chromatography
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Sephadex bead
Molecular sieve chromatography
20
Sephadex bead
Molecular sieve chromatography
21
Sephadex bead
Molecular sieve chromatography
22
Plain column of Sephadex Fancy column of Sephadex
23Handout 4-3: protein separations
Handout
24
Most chargedand smallest
Largest and most spherical
Lowest MW
Largest and least spherical
Similar to handout, but Winners &
Native PAGE added
Winners:
25
Enzymes = protein catalysts
26
g l u c o s e
monomers
MacromoleculesPolysaccharides LipidsNucleic AcidsProteins
biosynth
etic p
athway
intermediates
F l o w o f g l u c o s e i n E . c o l i
E ac h a rro w = a sp e c ific c h em ica l re ac tio nEach arrow = an ENZYME
27
H2 + I2 2 HI
H2 + I2 2 HI + energy
“Spontaneous” reaction:
Energy releasedGoes to the rightH-I is more stable than H-H or I-I hereThat’s why it “goes’ to the right, i.e., it will end up with more products than reactants
Chemical reaction between 2 reactants
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Cha
nge
in E
nerg
y (F
ree
Ener
gy)
H2 + I22 HI
{
-3 kcal/mole
2H + 2I
say, 100 kcal/mole
say, 103kcal/mole
Atom pulled completely apart(thought experiment)
Reaction goes spontaneously to the right
Energy change is negative: spontaneously to the right = exergonic: energy-releasing
Energy change is positive: spontaneously to the left = endergonic: energy-requiring
29
H2 + I2 2 HI
2 HIH2 + I2
2 HIH2 + I2
30
Cha
nge
in E
nerg
y (F
ree
Ener
gy)
H2 + I22 HI
2H + 2I
{
-3 kcal/mole
say, 100 kcal/mole
say, 103kcal/mole
But: it is not necessary to break molecule down to its atoms in order to rearrange them
31
H H
+I I
H H
IIII
H H
Transition state (TS) +H
I
H
I
(2 HI)
H H+I I
(H2 + I2)
32
Cha
nge
in E
nerg
y
H2 + I22 HI
2H + 2I
{
-3 kcal/mole
~100 kcal/mole
H-H| |I-I(TS)
ActivationEnergy
Say,~20 kcal/mole
33
Cha
nge
in E
nerg
y (n
ew s
cale
)
H2 + I22 HI
{
3 kcal/mole
Activation energy
HHII(TS)
Allows it to happen
determines speed = VELOCITY = rate of a reaction
Energy neededto bring molecules together to forma TS complex
Net energy change:Which way it will end up
DIRECTIONof the reaction, independent of the rate
34
3 glucose 18-carbon fatty acid
Free energy change: ~ 300 kcal per mole of glucose is REQUIRED
Biosynthesis of a fatty acid
3 glucose 18-carbon fatty acid
So getting a reaction to go in the direction you want is a problem(to be discussed next time)
35
Concerns about the cell’s chemical reactions
• Direction– We need it to go in the direction we want
• Speed– We need it to go fast enough to have the
cell double in one generation
– Catalysts deal with this second problem, which we will now consider
36
The catalyzed reaction
The velocity problem is solved by catalysts
The catalyst takes part in the reaction, but it itself emerges unchanged
37
Cha
nge
in E
nerg
y
H2 + I22 HI
Activation energywithoutcatalyst
HHII(TS)
TS complexwith catalyst
Activation energyWITH thecatalyst
38
Reactants in an enzyme-catalyzed reaction = “substrates”
39Reactants (substrates)
Not a substrateActive site
or substrate binding site
(not exactly synonymous,could be part of the active site)
40Unlike inorganic catalysts, Enzymes are specific
succinic dehydrogenaseHOOC-HC=CH-COOH <--------------------------------> HOOC-CH2-CH2-COOH
+2H
fumaric acid succinic acid
NOT a substrate for the enzyme:1-hydroxy-butenoate: HO-CH=CH-COOH (simple OH instead of one of the carboxyls)
Maleic acid
Platinum will work with all of these, indiscriminantly
41
Enzymes work as catalysts for two reasons:
1. They bind the substrates putting them in close proximity.
2. They participate in the reaction, weakening the covalent bonds of a substrate by its interaction with the enzyme’s amino acid side
groups (e.g., stretching).
+
42Chemical kinetics
Substrate Product (reactants in enzyme catalyzed reactions are called substrates)
S PVelocity = V = ΔP/ Δ t
So V also = -ΔS/ Δt (disappearance)
From the laws of mass action:ΔP/ Δt = - ΔS/ Δt = k1[S] – k2[P]
For the INITIAL reaction, [P] is small and can be neglected:ΔP/ Δt = - ΔS/ Δt = k1[S]
So the INITIAL velocity Vo = k1[S]
back reaction
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44
P
t
[S1]
[S2]
[S3]
[S4]
Vo = the slope in each caseVo = the slope in each case
Effect of different initial substrate concentrations
0.0
0.2
0.4
0.6
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Considering Vo as a function of [S](which wil be our usual useful consideration)
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