Bio 98 - Lecture 4

Amino acids, proteins & purification

Tryptophan and Tyrosine absorb UV light atlonger wavelengths than other amino acids

the molar extinction coefficient, is the optical density (OD) of a material at a given concentration, c, and l is the pathlength of the cuvette.

The molar extinction coefficient for Trp is 5,500 M-1 cm-1 at 280 nm, which means a 1 M solution of Trp has an optical density of 5,500 at 280 nm (OD280) when using a pathlength of 1 cm.

Lambert-Beer Law: OD or Abs = log10 Io/I = -log10 I/Io = c l

97.2%100%

Suppose we know that a protein contains 3 Trp and 4 Tyr residues. What is the extinction coefficient at 280 nm of the protein?

Extinction coeff. (1 M of substance) per Trp is 5,500 M-1cm-1 while that per Tyr is 1,400 M-1cm-1.

Total extinction coefficient of protein is then

nTrp x Trp + nTyr x Tyr = Total

3 x 5,500 M-1cm-1 + 4 x 1,400 M-1cm-1 = 22,100 M-1cm-1 =

So a 1 M solution of this protein has an absorbance (Abs or OD) of 22,100 at 280 nm (OD280).

What is the OD280 of a 10 M solution of this protein if we are using a cuvette with a pathlength of 1 cm?

xc x l = Abs280

22,100 M-1 cm-1 x 10-5 M x 1 cm = 0.221.

What fraction of the 280 nm photons have not been absorbed after passing through 1 cm of this sample?

Abs = log10 Io/I = -log10 I/Io or I/Io = 10-0.221 = 0.60

Isoelectric point (pI)

The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.

At a pH above their pI, proteins or amino acids carry a net ______ charge.

At a pH below their pI, they carry a net positive charge.

pI = 1/2 (pK1 + pK2) = 1/2 (2.34 + 9.60) = 5.97

AA with non-ionizable side chain (Gly)

pI = 1/2 (pK1 + pKR) = 1/2 (2.19 + 4.25) = 3.22

AA with ionizable side chain (Glu)

At a pH above their pI, proteins or amino acids carry a net negative charge.

Peptide: A-N-G-E-L-I-A

Ionizable group

pK Charge of group

protonated / de-protonated

Charge at pH 9.69

Charge at

pH 0

Ala (A)

C-term

2.34 0 / -1 -1 0

Glu (E)

side chain

4.25 0 / -1 -1 0

Ala (A)

N-term

9.69 +1 / 0 +0.5 1

Net charge -1.5 1

pI = (2.34+4.25) / 2

= 3.30

I. WHY PURIFY PROTEINS?

A. Research - academic

• to study function & regulation• to determine sequence & 3D structure• to clone and study the gene

B. Commercial ($$$) – practical

• research tools (restriction enzymes)• diagnostic reagents (prostate-specific antigen)• therapeutics (insulin, growth hormones, vaccines,

antibodies [biologics])

II. SOLUBLE vs MEMBRANE PROTEINS

• Proteins must be free in solution for purification. Not an issue for “soluble proteins.”

• Integral membrane proteins (those imbedded in the lipid bilayer) must be solubilized with a detergent first.

Membrane Protein ExtractionUsing Detergents

III. STEPS IN THE PURIFICATION OF A TYPICAL SOLUBLE PROTEIN

1. Homogenization: Prepare cell-free extract (rupture cell walls/membranes).

2. Centrifugation: Remove membranes, nuclei, large organelles (mitochondria) etc., keep supernatant.

3. Ammonium sulfate precipitation:(1) Proteins often become less soluble when the ionic strength is increased to very high levels. Precipitation points vary by protein, so purification can be achieved.(2) Precipitates can be redissolved in a small volume, so concentration can be achieved.(3) Dialyze the redissolved proteins against a low salt buffer.

4. Final purification with one or more rounds of Column Chromatography

Cell free extract

Add salt to 20% saturation

Centrifuge & remove

supernatantAdd salt to

40% saturation

Centrifuge & removesupernatant

Salt fractionation

Ammonium sulfate (NH4)2SO4

2NH4 SO4+ -2

Dialysis lowers salt concentration in a protein solution

before after

General Chromatography Protocol

OD280

Three Main Types of Chromatography

Ion (anion) exchange Size exclusion Specific affinity

Know Thy pI aka gel filtration

After [NaCl] increase, protein B will come off the bead before protein C…

Affinity purification of a genetically engineered (recombinant) protein containing an engineered purification tag

H

HH

H

Ni+5

His-tagged protein:Engineered, usually 6-His (HHHHHH) at N- or C-terminus

Affinity column or beads: immobilized Ni2+ or Co2+

How does one monitor the purification of a protein?SDS-PAGE (sodium dodecylsulfate - polyacrylamide gel electrophoresis)

About 1 SDS molecule binds per 2 amino acids

Activity vs. Specific Activity

Both beakers have the same activity (units) of ‘red’, though B has a higher specific activity as the ratio of red to other has increased.Specific activity = units/mg

A B

2D Gel (MW vs. isoelectric point)

Ribosome with and without L27 protein