Solubility

Chromatography Electrophoresis

Mr is the sum of the molecular weights of amino acids (-H2O) plus weights of other components. AAs have different Mr; 113/residue is a good average Volume can be estimated from the partial specific volume of the protein, which is between 0.72 and 0.75 mL/g for most proteins A protein of 200 residues is about 22,600 Da, or 3.8 x 10-20 g/molecule Its volume is about 2.8 x 10-20 cm3 or 2.8 x 104 3 Its radius is therefore about 19

Compound Glycine Lysozyme Trypsin Pepsin Horserdish peroxidase -Amylase LDH Catalase Glutamate dehydrogenase

Molecular Weight 33. Da 14. kDa 24 34 40.7 50 140 250 1,000

Semipermeable membrane Has a molecular weight cut-off Leads to selective dilution: Small molecules/ions diffuse freely They are diluted to the total volume (5:1005, in the example) Large molecules are trapped inside (unless the small molecules are 1. counterions to or 2. bound by the macromolecule)

1L
5 mL

Depends on interaction with solvent water Charge-dipole (depends on pH) Dipole-dipole H-bonding Proteins with >30% hydrophobic residues cannot cover them with polar groups, and must Associate with other proteins Associate with membrane Charge-charge attraction between proteins can decrease solubility (depends on pH)

Equilibrium

HA H+ + A-
+ or K A = [H ]

KA =

[H + ][A ] [HA]

[A ] [HA]

Take logs

logK A = log[H + ] + log

[A ] [HA]

[A ] + rearrange log[H ] = logK A + log [HA] Henderson-Hasselbalch equation pH = pK A + log [A ] [HA]

Charge vs pH
Imagine a polymer R with attached acidic group AH R-AH R-A- + H+ Net charge on the polymer follows the HH equation
0

pH = pK A + log

[A ] [HA]
H3N+-R-COOH H3N+-R-COO- H2N-R-COO-

-0.2

1.2 1 0.8

Charge

-0.4

0.6 0.4
Charge
1 2 3 4 5 6

-0.6

0.2 0 -0.2

-0.8

-1

-0.4 -0.6 -0.8 -1

pH

[A ] pH = pK A + log [HA]

5
pH

11

Example: 22 Acidic Groups and 12 Basic Groups uniform pKas

15 10 5 0
Charge

Guessing the Isoelectric Point 22 Acidic Groups and 12 Basic Groups

More Acidic groups - so, pI will be acidic Which means all basic groups will be protonated (+) You must deprotonate enough acidic groups to balance them:

Isoelectric point (pH) Positively charged

1 2 3 4 5 6 7 8 9 10 11 12

-5 -10 -15 -20 -25

pH

12+ charges (Lys + Arg sidechains, N-terminus) you need 12 - charges (-COO-) leaving 10 protonated -COOH

Negatively charged

pI = pH = pK a + log

12 10

= 4 + .08 4.1
Better: use Excel with real pKas

Isoelectric Point
Proteins are LEAST soluble at their isoelectric point No net charge - minimal repulsion Perhaps +/- local attractive charge interactions Proteins with different isoelectric points MAY be separable by adjusting the pH Is NaCl equivalent to (NH4)2SO4? First, dene concentration of salt solution, Ionic Strength 1 I = mi Z 2 i 2 where mi is the molal concentration of component i and Zi is the charge on component i 1M NaCl 1m) 1M Na+ plus 1M Cl- ( 112 + 1 (1) 2 I= = 1M 2 + plus 1M SO 2- 1M (NH4)2SO4 2M NH4 4
2 12 + 1 (2) 2 I= = 3M 2

Complication for weak acids or bases Ionic Strength depends on pH:

[H2CO3] + [HCO3-] + [CO32-] = 0.20 M At pH 10, [H2CO3] 0, so [HCO3-] + [CO32-] = 0.20 M

pH = pK a + log [CO 2 ] 3 [HCO ] 3 10 = 10.3 + log [CO 2 ] 3 [HCO ] 3 [CO 2 ] 3 = 0.5 [HCO ] 3

H2CO3 H+ + HCO3- H+ + CO32-

Use the H-H equation to Calculate [HCO3-] and [CO32-]

0.2M = [HCO3-] + [CO32-] = [HCO3-] + 0.5 [HCO3-]

Z = 0, doesnt matter Z = -1 Z = -2 [H+] is usually negligible

[HCO3-] = 0.13 M and [CO32-] = 0.07 M

So, per liter, 0.13 moles of NaHCO3 and 0.07 moles of Na2CO3
I= 1 {[Na + ](+1) 2 +[HCO ](1) 2 + [CO 2 ](2) 2 } 3 3 2

Enzyme 2
log (solubility)

Enzyme 3

Enzyme 1 20% 40% 60% 80%

Ammonium sulfate (% of saturation)

Proteins may associate by charge-charge interactions Less interaction with solvent means lower solubility The charge-charge force is described as
Forcevacuum = Z + Z r2 Z Z Dr 2
+

in a vacuum, or in a solvent of dialectric constant D

A stationary phase A mobile phase Solutes that partition between them

Forcesolvent =

For Water, D >80 For EtOH, D = 35 Mixtures, in between proteins may precipitate Use any water-miscible solvent

Systems
Mobile Gas Gas Liquid Liquid Liquid Stat Solid Liquid Solid Liquid Liquid Method GC (fract., distil.) G(L)PC (capillary) LSC (silica, alumina) Partition LC (RP) countercurrent distribution

General Theory- Definitions

Partition Coefficient Phase Ratio
K= CS CM VM VS

Capacity Ratio

k=

K CS VS = C M VM

Amount of solute

As = CsVs

General Theory- Ave.Speed

Ave. Speed of Solute
S = SM f M + SS f S = SM f M

General Theory- Ave.Speed

Rearrange:
S 1 = = Rf SM 1+ k
TLC or Paper

Fraction of time in mobile phase

AM C MVM 1 1 = = = AM + AS C MVM + CSVS 1+ CS VS 1+ k C M VM

1 S = SM 1+ k

So,

If solvent & solute travel for the same length of time, speed distance. So, measure:

Rf =

Spotdistance Solventdistance

Column Chromatography
Solvent and solute must travel the same distance (in different times) So, invert everything, measure retention time, tr, or elution volume, Ve

Play with the Equation

Ve = 1+ k VM Substitute for k: Multiply by VM
Ve CV = 1+ S S VM C MVM
Only works if you can measure (or define) VS i.e., partition chromatography Always works

t r Ve SM = = = 1+ k t m VM S
(At constant flow rate t V chart distance)

CV Ve = VM + S S = VM + KVS CM

Solve for K or k
Different for every peak

Problem: predicts very sharp peaks

Try another approach: model the process as a sequential extraction 1. Put solvent (stationary phase) in tubes 2. Put solute in first tube only 3. Put immiscible solvent in first tube 4. Shake, solute distributes itself according to K
CM CS

k=

Ve 1( duh ) VM

K=

Ve VM VS

Only if Vs is defined Not adsorption chromatog

Sequential Extraction
Transfer only the upper phase to the next tube

Sequential Extraction
Transfer only the upper phase to the next tube

Fill first tube with fresh upper solvent

Solute that prefers the mobile phase travels faster Model the process as material balance about the nth tube as small volume dv is transferred Amtn = Amt in - Amt out = Cn-1dv-Cndv

Shake, repeat

Equations

Cn as function of volume flowing

For each Cn, you have to know Cn-1, so you have to calculate ALL previous Ci Luckily, a pattern develops:
Cn = Ci0 (aV ) ni aV e (n 1)! i=1
n

Ci = conc of solute in the mobile phase of tube i

Amtn = Amt in - Amt out = Cn-1dv - Cndv But the change in total amount = d[CnVm+ CsVs] or, Cn-1dv - Cndv = d[CnVm+ CsVs] Substitute,for Cs using K = Cs/Cn Cn-1dv - Cndv = d[Cn(Vs + KVs)] = (Vs + KVs)Cn Rearrange:

0 Also, all C 0 = 0 except for C1 i

dC n Cn C n1 + = dV VM + KVS VM + KVS

Cn = Ci0

(aV ) ni aV e (n 1)!

Plot Cn vs volume that flows

Cn = Ci0 (aV ) ni aV e (n 1)!
Cn vs tube # as solvent flows
0.4 0.35
Concentration in tube n

Interpretation
Make a simpler variable a = 1/(VM + KVS) Volume is in units of 1/a On a TLC plate, as solvent flows
Solute Spots

V=1 V=5 V = 10 V = 20

0.3 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 Tube number 25 30 35 40

moves along the plate broaden If different solutes have different K, they move at different rates (distances)

Column Chromatography
Dont care how solutes are distributed on the column, but when (at what volume) they come OUT We need to know the solute concentration in the LAST tube, and how that changes with the volume that flowsa chromatogram

Chromatogram
Let the number of tubes N be a very large number (>100), so that N N-1 Calculate CN

CN = C0 i

(aV) N aV e (N)!

or

C N (aV) N aV = e C0 (N)! i

Chromatogram
C N (aV) N aV = e C0 (N)! i
0.045 0.04 0.035 0.03
C in last tube

Who cares?

We do

What is the elution volume? What controls the width?

For Ve, take first derivative, set to zero, solve for V

0.045 0.04 0.035
C in last tube

0.025 0.02 0.015 0.01 0.005 0 50 70 90 110 130 150 V (multiples of 1/a)

I simplified to

Y = (eX )

X N!

0.03 0.025 0.02 0.015 0.01

And used d(uw) = udw + wdu = 0 I got X = N, or aV = N or

0.005 0 50 70 90 110 130 150 V (multiples of 1/a)

N Ve = = N(VM + KVS ) a

Where the Vs are for each tubemultiply by the # of tubes.

Who cares?
Ve is the same as for the simple treatment What else do we learn? Width?

Width at Base?
Get the Ls from the slope of tangent (rise over run = derivative)
C in last tube

0.045 0.04

Take derivative & set to zero Draw tangents thru the inflection points
0.045 0.04 0.035 0.03
C in last tube

2nd

0.035 0.03

0.025 0.02 0.015 0.01 0.005 0 50

Yinflection

Yinflection

Add the four segments: Width = aV= L1 + (N-Xinf) + (Xinf-N)+ L2 Get L1 = N 1 L2 = N + 1 So, width = aV = 4 N
0.02 0.015 0.01 0.005 0 50

0.025

Yinflection

Yinflection

L1
70 90

L2
N
110 130 150

V (multiples of 1/a)

X inflection = N N

N + N = X inflection

L1
70 90

L2
N
110 130 150

V (multiples of 1/a)

X inflection = N N

N + N = X inflection

We also know that aVe = N, so V 2 aVe N N N = 16 e = = or V aV 4 N 4

Calculate the number of Plates

From the elution volume and width at base (in the same units), vol, time, chart distance 2 More plates means less broadening per unit flow, higher column efficiency Theoretical Plates or Plate Number
V N = 16 e V

Who cares?

We do Resolution depends on having different Ve, but also narrow lines

Ve2

R=

2(Ve ) W1 + W2

Ve1

V 2 N = 16 e W

N is important
W1 W2

What determines N?
Unfortunately, Plate Theory is a lie. The solute never reaches equilibrium There is continuous flow It is the competition between partitioning and flow that matters Use Rate Theory - Giddings Too hard for me

The Giddings Equation

L/H is the height equivalent to a theoretical plate, H or HEPT

1 1 1 + C edp Cm dp 2 / Dm

+

H=

C dD m Csm dp 2 Cs df + + Dm Ds

, linear solvent velocity dp, particle diameter ds, thickness of stat phase film Di, diffusion coeffs, s and m phases

. eddy currents . mass transfer in m phase . longitudinal diffusion . stagnant m phase escape . mass xfer in stat phase

Lessons from Giddings

Longitudinal diffusion-limited Mass transferlimited

Lessons from Giddings - 2

Smaller column packing Larger packing
Stagnant stationary phase

HETP

optimum solvent velocity

reasonable working range Solvent velocity

Biphasic behavior

Different flow paths

Different solvent velocities Stagnant mobile phase

There is an optimum gas velocity Probably always above opt velocity for liquids, so usually you can increase resolution by decreasing the flow rate

Smaller particles are always better Remove particles altogether for best performance (capillaries)

A stationary phase A mobile phase Solutes that partition between them

Pour a column

Remove buffer and layer the sample on top

Let sample enter collect or measure eluate layer buffer on top

Attach to resevoir

For all kinds of chromatography, retention time: or or

[Protein] (mg/mL) Activity (U/mL)

250

tr = t o + k t o

tr = 1+ k to

Ve = 1+ k Vo

Protein Activity
200

[solute]

Ve 2 Ve 1 time or volume

Ve 3

150

100

50

If the volume of stationary phase is dened:

Ve = Vo + K d Vs
0

50

100

150

200

250

Volume (mL)

Column is packed with porous beads Vbed is the volume of packing Vs is the volume of the beads Vo is between beads (void volume) Vs Vo Bed height Vbed= r2 (bed height)

Column is packed with porous beads Small molecules can enter pores, large molecules cannot

Enters pores freely Ve = Vo + 1Vs Enters partially Ve = Vo + KVs Does not enter Ve = Vo

Vo = volume between beads Vs = volume of the beads, including pores Vbed = Vo + Vs so Vs = Vbed - Vo Measure Vbed physically, or calculate it geometrically Measure Vo as Ve of a very large molecule (blue dextran)
Ve = V0 + K av Vs

Cytochrome c Hemoglobin BSA AdH Catalase Urease Ferritin

K depends on apparent molecular size, not Mr Assume hemispherical pores, with volume of

av

0 3 4

Fibrinogen

5 Log Mr

2 vol = r 3 3

V Vo V Vo K av = e = e VS Vbed Vo

Kav

approximate correlation

Large molecule of radius a cant t as easily. The pore appears to have a 2 volume of

volapp = (r a) 3 3
a

Log (Mr)

Ve = V0 + K VS

K is actually the same for all solutes, K = 1 Different sized molecules see pores differently Lets pretend K changes
K app V pore = K true Vapp 2 Vapp V (r a) 3 (r a) 3 = 1 K true app = 3 2 = Solve: K app = K true V pore V pore (r) 3 (r) 3 3

app

(K )

1/ 3

vs a should give a straight line

1/3

This a is the Stokes radius, Rs from f = 6Rs

K K(1/3)

Desalting Removal of substrate or inhibitor Change buffer Separate Proteins of different size Estimate molecular weight Kav Log Mr (if standard and unknown have the same shape) Estimate Stokes radius (apparent hydrodynamic radius) Several equations exist

20

40

60

80

100

Rs

()

Stationary Phase (column packing) Sephadex or Sepharose Biogel P or Biogel A (polyacrylamide or agarose) TSK (coated silica) Resins (porous poly(styrene/divinyl benzene)) Mobile phase Aqueous buffers of reasonable ionic strength Systems exist for Organic solvents Denatured proteins separates subunits removes shape ambiguity Many molecular size ranges

Cl + - + - Cl- ClCl- + - Cl Cl- + + Cl + + Cl- Cl ClCl- Cl + + ++- Cl- +- +Cl- + Cl Cl +Cl++ -Cl+Cl- +- Cl -+Cl-+Cl- - +Cl Cl+- Cl Cl + + Cl+ Cl+ClCl++ + Cl+ +Cl- Cl Cl+ Cl Cl Cl +Cl-+Cl- +Cl- +Cl- + - +- +- + - + Cl + Cl +- Cl++ -++- Cl+ -Cl ++Cl- +- Cl+ Cl + Cl- Cl - + Cl + Cl - Cl Cl- + Cl + + Cl + + - ClCl-Cl- ++Cl- -+ - Cl- Cl- + ClCl + +Cl-+ +Cl Cl + -+Cl- ClCl- +- Cl- + - + ClCl + ClCl +Cl- +-+Cl- + -+Cl- +Cl-Cl + + +-Cl Cl + - + Cl- Cl + + + Cl Cl ClClCl-

Cl-

Beads have charged groups on the surface. Charged groups are paired with counter-ions A protein with surface charges opposite to those on the bead surface can bind to the bead by displacing the counterions. More charge better binding

ClClClClClClCl- Cl ClClClCl- Cl- Cl- ClClCl- Cl- Cl - Cl- Cl- Cl- Cl Cl- Cl ClClCl Cl- ClClClCl- ClClCl- Cl Cl- Cl- Cl-ClCl- ClClCl Cl ClCl- Cl ClCl- ClClCl ClCl- Cl ClCl- ClClCl- ClCl- Cl- ClCl Cl- Cl Cl- ClClCl- Cl -Cl Cl Cl ClClCl- ClCl Cl-

++ + + + + + +++ + + + ++ + + + + + + + + ++ + + + + + + + + ++ ++ + ++ + + ++ ++ + +++++ + + + + + + + +++ ++ + + + + + + + + + + + ++ + + + + + + + + + + + +

Cl-

Cl-

ClClClCl-

ClCl-

1. Load column Ionic strength is low pH is correct above or below pI so k is large for the protein of interest 2. Wash column with loading buffer or similar (dilute salt) 3. Elute Isocratic (usually not, only for weak binding) Batchwise (increasing xed [NaCl]) Linear gradient of NaCl 4. Regenerate column packing
[Protein]

Variety

Examples

[Na

Cl]

load

wash

elute

Anion Exchange Tertiary Amines DEAE-Cellulose (Diethylaminoethyl) DEAE Sephadex Quaternary Amines TriethylaminoQAEPolyethylene PEI imine Cation Exchange Carboxymethyl CM-Cellulose CM-Sephadex Arylsulfonate (rare for proteins)

No charge, wrong charge, elute at Vo

Vol

Increasing charge density

Inert phase is typically cellulose, Sephadex, silica, coated silica (TSK), resins (PSDVB) Buffers

Calcium Phosphate Hydroxyapatite

10

Commonly use sepharose or Sephadex Activated with CNBr

OH OH N C Br O C OH

O O

C NH + H2N R X

O O

C N

R X O C O NH R X

+ OH

H2N(CH2)6NH2 and H2N(CH2)6CO2H Are common spacer arms

Bead-N(CH2)6NH2 is a nucleophile and can be attached to many biochemicals (e.g., 8-Br-AMP) or to proteins by making amide bonds to Asp & Glu sidechains (using carbodiimide reagents) Bead-N(CH2)6CO2H can be attached to amines, including proteins, by making amide bonds (to Lys sidechain, using carbodiimide reagents) Several other spacer arms are common

1. Load the column, low salt, etc. Most proteins do not bind Stop loading if activity starts to pass through 2. Wash with loading buffer to remove un-bound and nonspecically-bound protein 3. Elute with competitor (second chance at specicity)
[Activity] [Protein]

load

wash

elute

11

We understand why an enzyme might bind immobilized AMP But some enzymes bind immobilized aromatic dyes (e.g., Afgel Blue) even though they do not resemble substrate or inhibitor otherwise, behave just like afnity ligands Examples (on sepharose, agarose or cellulose) Cibacron Blue 3GA Reactive Green-19 Reactive Red-120 Buy a set of immobilized dyes and screen for binding, elution

Attach marcomolecules as a stationary phase Antibodies Lectins e.g., Concanavalin A Avidin/Steptavidin Inhibitor/antagonist e.g., egg ovomucoid trypsin inhibitor Or immobilize metal ions (e.g., Ni2+) by chelation chromatograph His-tagged proteins

Began as control experiments for Afnity Chromatog. Commonly used stationary phases Butyl sepharose or agarose Octyl sepharose or agarose Phenyl sepharose or agarose Use Ammonium Sulfate to decrease solubility of the proteins Elute with gradient of amm. sulf., & gradient of eth. glycol Hydrophobic hairs on bead protrude into hydrophobic pockets polar surfaces interact, also
Bead
C O NH C H2 O C NH O C NH2 C NH2

t u b e

s l a b
ho

riz

on

ta

Free-radical polymerization leads to a cross-linked gel Acrylamide concentration determines overall density of gel Bis- methyleneacrylamide determines the extent of crosslinking Gels are characterized by %T and %C
%T = [grams of (acrylamide + bis) / volume (mL)] x 100 %C = [grams of bis / grams of (acrylamide + bis)] x 100

Casting gels (free radical polymerization)

O O O N C H2 N H H C C H C H2

H2N

O O

C H2 C H C

NH2

+ m C H2

CH C

acrylamide

N,N-methylene-bisacrylamide

H2N

Total acrylamide (approximately % by wt) 5% < T < 20% Determines pore size Crosslinker (% of acrylamide that is bis, by wt) 2% < C < 5% Determines tensile properties

12

1. 2. 3. 4. 5.

Polymerize gel in tube or between glass plates Put samples in wells of gel, include tracking dye Suspend gel between two buffer reservoirs Apply constant voltage (100V) Turn off electricity when tracking dye approaches the bottom of gel 6. Remove gel from tube or plates 7. Fix and stain (methanol/acetic acid/water, Coomassie Blue) xing - methanol/acetic acid precipitates the proteins Coomassie Blue stains everything 8. Destain (methanol/acetic acid/water)

Ion with Z charges of e units in an electric eld E F FE = ZeE feels a force and accelerates a = mass Particle with frictional coefcient f moving with velocity V F f = f V feels a frictional force (drag) Acceleration stops and the particle moves at constant speed when FE = Ff or ZeE = Vf Which can be re-written as V Ze velocity charge on protein = E f unit field strength friction (size)

Separation depends on charge Can vary the charge by changing the pH Separation depends on size Cant vary size, but you can Vary frictional drag by increasing %T Analogy to Stokes eqn.: f = 6Rs, (%T ) At constant pH, can vary %T and plot mobility vs %T
Log (mobility)

Intercept depends on charge (pH)

Slope depends on size

largest least charged

10

15

20

%T

Remove uncertainty about Shape, by denaturation Charge, by coating protein with ionic material O Sodium dodecyl sulfate (and heat) O S Unfolds the protein O randomizes the shape, so f size Coats hydrophobic portions provides negative charge larger proteins bind more, so acceleration = FE /mass is the same for all proteins Mobility depends only on friction, which depends on Mr Include 2-mercaptoethanol to reduce disulde bonds Beware that glycoproteins bind less detergent Lower acceleration, therefore Appear larger

13

Mobility depends only on size (not shape or charge) Mobility depends only on size (not shape or charge) Plot the mobility vs Log (Mr) (Ferguson Plot)
Mobility (cm or % of tracking dye)

Standards

Unknown

Log (Mr)

Same experiment, but pour the gels with a gradient maker

pH 11

Low %T

High %T

pH 2-3

+
Pour gel containing

+
Proteins behave like ampholytes Distribute according to their pI

ampholytes, a mixture of amphoteric amines/ acids

Use acidic buffer at bottom, alkaline at top Negative ampholytes move toward anode until they become protonated, stop at pI

Run IEF gel in one dimension Treat with SDS Place treated IEF gel on top of SDS gel Electrophorese into SDS Gel to get a 2-Dimensional Separation pI

Monomer Mr

1. Coomassie Brilliant Blue R in methanol/acetic acid water destain in similar solution without dye scan with densitometer 2. Colloidal gold or silver 10x more sensitive 3. Activity stains - very selective Infuse substrates into gel after electrophoresis colorimetric (UV/Vis) indicator Zymogram e.g., look for clarication of starch gel 4. Antibody stain (Western Blot)

14

AcSCoA + Pi

AcP + CoASH
A600 A412
0 2

Stain with Ellmans Reagent (DTNB - Stains Thiols) Scan at 412 nm Fix & stain with Coomassie Scan at 600 nm The phosphotransacetylase is a minor component The sample is mostly another protein!

Mobility (cm)

15