1. Bio 98 - Lecture 4 Amino acids, proteins & purification

2. Tryptophan and Tyrosine absorb UV light at longer 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 LawLambert-Beer Law: OD or Abs = log 10 I o /I = -log 10 I/I o =  c l 97.2%100%

3. 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 -1 cm -1 while that per Tyr is 1,400 M -1 cm -1. Total extinction coefficient of protein is then n Trp x  Trp + n Tyr x  Tyr =  Total 3 x 5,500 M -1 cm -1 + 4 x 1,400 M -1 cm -1 = 22,100 M -1 cm -1 =  So a 1 M solution of this protein has an absorbance (Abs or OD) of 22,100 at 280 nm (OD280). 

4. What is the OD280 of a 10  M solution of this protein if we are using a cuvette with a pathlength of 1 cm?  x  c x l = Abs 280 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 = log 10 I o /I = -log 10 I/I o or I/I o = 10 -0.221 = 0.60

5. 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 (pK 1 + pK 2 ) = 1/2 (2.34 + 9.60) = 5.97 AA with non-ionizable side chain (Gly) pI = 1/2 (pK 1 + pK R ) = 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.

6. Peptide: A-N-G-E-L-I-A Ionizable group pKCharge of group protonated / de- protonated Charge at pH 9.69 Charge at pH 0 Ala (A) C-term 2.340 / -10 Glu (E) side chain 4.250 / -10 Ala (A) N-term 9.69+1 / 0+0.51 Net charge-1.51 pI = (2.34+4.25) / 2 = 3.30

7. 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])biologics

8. 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.

9. Membrane Protein Extraction Using Detergents

10. 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: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

11. Cell free extract Add salt to 20% saturation Centrifuge & remove supernatant Add salt to 40% saturation Centrifuge & remove supernatant Salt fractionation Ammonium sulfate (NH 4 ) 2 SO 4 2NH 4 SO 4 +-2

12. Dialysis lowers salt concentration in a protein solution beforeafter

13. General Chromatography Protocol OD280

14. Three Main Types of Chromatography Ion (anion) exchangeIon (anion) exchange Size exclusion Specific affinitySize exclusionSpecific affinity Know Thy pI aka gel filtration

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

19. Affinity purification of a genetically engineered (recombinant) protein containing an engineered purification tag H H H H Ni +5 His-tagged protein: Engineered, usually 6-His (HHHHHH) at N- or C-terminus Affinity column or beads: immobilized Ni 2+ or Co 2+

20. 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

22. 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

23. 2D Gel (MW vs. isoelectric point) Ribosome with and without L27 protein