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

7

centrifugal 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 charge

Loser:Large, +low positive charge

Intermediate:Large, +++high positive charge

Intermediate: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)

16

Summary 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

19

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

biosy

nthet

ic p

athw

ay

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

28

Ch

ang

e in

En

erg

y (F

ree

En

erg

y)

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

Ch

ang

e in

En

erg

y (F

ree

En

erg

y)

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

Ch

ang

e in

En

erg

y

H2 + I22 HI

2H + 2I

{

-3 kcal/mole

~100 kcal/mole

H-H| |I-I(TS)

ActivationEnergy

Say,~20 kcal/mole

33

Ch

ang

e in

En

erg

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

Ch

ang

e in

En

erg

y

H2 + I22 HI

Activation energywithoutcatalyst

HHII(TS)

TS complexwith catalyst

Activation energyWITH thecatalyst

38

Reactants in an enzyme-catalyzed reaction = “substrates”

39

Reactants (substrates)

Not a substrate

Active site or

substrate binding site(not exactly synonymous,

could be part of the active site)

40Unlike inorganic catalysts, Enzymes are specific

                                     succinic dehydrogenase

HOOC-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 P

Velocity = 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

43

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

45

Considering Vo as a function of [S](which wil be our usual useful consideration)