Chapter 11 Lecture Elastic and Periodic Motion, Study notes of Physics

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Protein Purification

September 14, 2023

Lecture Notes

2 Understand what is necessary for protein isolation Be able to think about the strategy of protein isolation Understand the different types of chromatography Understand what gel electrophoresis is and what results you can get

1. Protein Purification

4

Protein Purification

The ability to understand the biological process parallels our ability to isolate biomolecules. A biological substance may only be 0.1% of a cell mass. It must be purified to over 98% pure by isolation procedures before one can study its chemistry.

Compositions of Some Proteins

Methods of solubilization of animal cells

Cells can be lysed by hypotonic shock. Cells with high salt inside and no salt outside will swell and rupture Bacteria outer membranes must be digested. Gram-negative bacteria

• Hen egg white lysozyme digests b (1-4) linkages in the (glycosidic bonds) of polysaccharides. Mechanical breakage blenders homogenizers
• French press - high pressure 20,000 lbs/in 2 forced through a small hole disrupts cells
• ultrasound or sonication disrupts cells.

Stability: Proteins can denature

H-bonds, ionic bonds, Van der Waals interactions, and Hydrophobic interactions can be disrupted. Denaturation is the process by which a protein loses its “native” or active shape or conformation. Temperature can play a role “cold labile” “heat labile” Protect against-Proteases, Inhibitors, Changes in pH, Protein can be air-denatured -egg white meringue - absorption to surfaces Damaged by oxidation O 2 Heavy metals damage proteins -they bind to protein- Cu

Hg

Bacterial contamination can destroy the protein

Activity Measurements

In order to follow the purity of an enzyme, you need a method

to measure its activity.

Spectrophotometric analysis- is one common method to measure activity. Substrate [S] Product [P] a change of [S] with time if S is colored “absorbs light” we can use Beer’s Law. A = eb c c - concentration e - millimolar extinction coefficient A - absorbance b - path length T - percent transmittance A = - log % T if  A then  c at  max

Start with one liter of lysed cells.

We measure the rate of .01 ml of cells at at concentration of 20 mg/ml. i.e. the amount of enzyme we will assay is 0.01 ml We get a rate of  A = 0.5 A/min 1 millimolar = 6.22 = e mM 0.5/6.22 = .08 millmolar/min and our assay volume = 1 ml 1 millimolar in a volume of one ml = 1 micromole/ml = mole C=.08 moles in 1 ml/min = .4 moles 0.2 mg min/mg

Example total activity versus specific activity

Total activity: .04 moles x 20 mg/ml = 0.8 moles / ml 0.8 moles x 1000 ml = 800 moles in 1 liter of cells ml min Red = is our enzyme If we remove greens & blues the specific activity increases, however, our total activity remains the same. If We lose red the total activity decreases.

Some enzymes have no easy assay but the product of the reaction can be used in another reaction: Coupled Reactions: We couple enz 2 to enz 1 and measure NADH to get A enz 1 enz 2 A B C NADH NAD

Use of Radioactivity

ATP  ADP + Pi Separate ATP + Pi + ADP on TLC measure radioactivity Phosphoimager makes this easy else cut spots and count in scintillation counter. N N N N NH 2 O OH H H H H H -O P O O- O -O P O O- O -O P O-O O- O Pi ATP

Strategy Continued

Characteristic Procedure •Size 1. Dialysis and ultrafiltration

2. Gel electrophoresis
3. Gel filtration
4. Ultracentrifugation •Specificity 1. Affinity chromatography
5. Immunopurification •Solubility 1. Salt precipitation
6. Detergent solubilization

Ionic Strength

Ci = the molar concentration of the ith species Zi = it’s ionic charge 1M Na

Cl

• Z = 1 Na + Z = 1 Cl

1 = (1M x 1)Na + (1M x 1)Cl 2

i

c Z

I

i