1. C2-3. Page 144 Table C.2-3. Lactate Dehydrogenase Reaction Time Courses Reading number time (seconds) A 340 readings 50 µl sample 100 µl sample 200 µl sample 300 µl sample 400 µl sample 10 215 330 445 560 675 790 8105 9120

2. Protein Purification Lab C2 Pages 115 to 168 Lab C.2 Four Periods Protocol Page 135-160 Benchtop Protocols begin on page 398 Be sure to read theory starting page 120

3. Exam Exam October 19,20 Includes Carbohydrates, Enzyme kinetics, and all protein labs and material related there to. Pay attention to the powerpoints –Read theory sections in the lab manual Will be about one hour in length Example of exam with answers is posted on web

4. This Lab 4 lab periods Prelab= 12 points Lab Report= 55 points First exam in period 4

5. You Have: Become skilled at using micro pipetters Have learned to use the spectrophotometer –To determine concentration of an unknown Beers Law –To measure activity of an enzyme Have learned how to organize experimental protocols Have learned how to prepare a report.

6. In the next days You will use all of these skills to perform a fundamental exercise in Biochemistry/Molecular Biology Will learn basic protocols in protein purification and analysis

7. Protein Purification A black art (proteins have personality) Requires knowledge of protein –What kind of cell is it coming from –What part of cell –What does it do Particularly helpful –Size –Composition

8. Strategy Move from organism to pure protein in as few steps as possible with as little loss of activity (assayable quality) as possible –Time and temperature are factors

9. Next 4 sessions Day one: Protein fractionation by centrifugation, salt precipitation and dialysis Day 2: Purification by affinity Chromatography Day 3: Determination of concentration by BCA assay Day 4: Determine purity by PAGE

10. Will fill out this critical table as we proceed page 162 (day 4) Table C.2-4. Enzyme Purification Table Net volume (ml) V 0 units per ml V 0 units Total (an “amount”) Protein content (% of total) Protein concentration (mg/ml) Net amount of protein (mg) Specific Activity (V 0 /mg protein) StepABCDEFG 1. Cleared 2. (NH 4 ) 2 SO 4 Supernata nt 3. diluted dialyzed sample/ solution placed on column 4. pooled peak tubes from column Column C = (Column A)(Column B) Column F = (Column A)(Column E) Column G = Column C/Column F = Column B / Column E Column D = Column C/first value in Column C

11. Protocols for Protein Purification Highly individualized Use a common approach –Fractionate crude extract in a way that protein of interest always goes into the pellet or the supernatant. –Follow progress with functional assay

12. Lactate Dehydrogenase NADH + H + + Pyruvate  =  NAD + + Lactate Enzyme clears lactic acid from working muscles The obvious source of enzyme is muscle tissue (heart & skeletal muscle, H&M, isomers) We will assay for the enzymes ability to convert Pyruvate to Lactate

13. Begin with intact tissue Disrupt (step4&5) –Blender, homoginizer Remove debris (step7) –Centrifugation Precipitate/concentrate (step 14-16) –Ammonium sulfate Remove salt (step 22) –dialysis Purify (next Lab) –Chromatography Analyze (Part B and week 3 & 4) –Activity, molecular weight

14. Ammonium Sulfate ppt page 124 Has a wide range of application Relies on fact that proteins loose solubility as concentration of salt is increased –Is characteristic of particular protein –Results in a partial purification of all proteins with similar solubility characteristics –Must determine [amm sulf] to precipitate your protein empirically. Produces “salt cuts”

15. Salting in / Salting out Salting IN At low concentrations, added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions. So low [salt] prevents aggregation and therefore precipitation or “crashing.” Salting OUT At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H 2 O) needed to solvate the macromolecules. So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

16. Kosmotrope vs. Chaotrope Page 125 Ammonium Sulfate Increasing conc causes proteins to precipitate stably. Kosmotropic ion = stabilizing ion. Urea Increasing conc denatures proteins; when they finally do precipitate, it is random and aggregated. Chaotropic ion = denaturing ion.

17. Dialysis Page 180 Passage of solutes through a semi-permeable membrane. Pores in the dialysis membrane are of a certain size. Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

18. Column Chromatography 2 nd Day Page 126

19. Available in any volume

20. Gel Filtration

21. Principles of gel filtration (molecular sieving) 10 6 Da 3x10 5 Da 10 5 Da 10 4 Da 1. Apply a mixture of proteins on a gel filtration column (Sepharose, Sephacryl, etc) 2. Collect fractions, typically 120 from a 1.5x100 cm column. Do not change buffer composition 3. High molecular weight macromolecules (higher Stoke’s radius) elute first 4. Determine proteins in eluate using suitable assay 5. Estimate approximate molecular weight of unknown proteins and/or protein complexes using calibration curve with pre-run standard proteins of known M.Wt. and the following formula: Kav = Ve -Vo Vt - Vo Ve – elution volume Vo – void volume Vt – total volume Kav Log M.Wt.

22. Ion Exchange

23. Affinity Chromatography We will use bound Adenosine -5’-monophosphate (page 128 & 152). This is part Of NAD +. LDH will Bind. Release LDH by adding NADH

24. NAD+ AMP

25. Affinity chromatography Remember: NADH is a co-substrate for lactate dehydrogenase. We use AMP-Sepharose: AMP is covalently bound to the affinity gel, which will not pass through the filter. LDH binds to the AMP b/c it looks like half an NADH. Thus LDH remains immobilized in the column until we add NADH which binds tighter to the LDH.

26. Protein Purification page 130 A 280 Activity NADH

27. Protein Concentration Lowry ( most cited reference in biology) –Color assay A 280 –Intrinsic absorbance Page 132 –Relies on aromatic amino acids BCA page 133 –Modification of Lowry: increased sensitivity and consistency Bradford –Shifts A max of dye from 465nm to 595nm

28. A 280 Page 132 Uses intrinsic absorbance Detects aromatic residues –Resonating bonds Depends on protein structure, native state and AA composition Retains protein function

29. Protein separation using SDS-PAGE (Laemmli system) Stacking gel Resolving gel 1. Apply protein/dye samples into polyacrylamide gel wells 2. Run the electrophoresis until dye reaches the end of the gel 3. Remove the gel from the apparatus and stain for proteins Page 158

30. SDS PAGE of Purification Process 1.Complete mix of proteins 2.High Salt 3.Ion exchange 4.Gel-filtratio 5.Affinity 10micrograms loaded in each lane

31. IMPORTANT Do not throw away anything until you are certain you no longer need it –Biggest source of problem in this lab Label everything clearly copy labels into lab book Throwing out wrong fraction results in starting over –3 days into experiment huge problem

32. Day 1: See Table C2-2 (page 117), Page 118- 124. & 138

33. Will follow Flow sheet: Page 138 We will do only one NH4SO4 cut Save 3 samples Will determine protein concentration activity and purity

34. Will fill out this critical table as we proceed page 162 Table C.2-4. Enzyme Purification Table Net volume (ml) V 0 units per ml V 0 units Total (an “amount”) Protein content (% of total) Protein concentration (mg/ml) Net amount of protein (mg) Specific Activity (V 0 /mg protein) StepABCDEFG 1. Cleared 2. (NH 4 ) 2 SO 4 Supernata nt 3. diluted dialyzed sample/ solution placed on column 4. pooled peak tubes from column Column C = (Column A)(Column B) Column F = (Column A)(Column E) Column G = Column C/Column F = Column B / Column E Column D = Column C/first value in Column C

35. Today. Page 138 (part of group) Steps 1-5: Weigh muscle sample place in blender with 50ml ice cold buffer homogenize for 2 minutes. Steps 6&7: remove large debris by centrifugation Save Supernatant (remove 1ml (Microfuge tube) for later analysis). Steps 9-13: Measure the volume of the supernatant determine amount of ammonium sulfate required for precipitation, weigh out 0.4 grams per/ml (NH4) 2 SO 4

36. Today group 1 continued Step14-16: Slowly add salt to gently stirred supernatant. Keep Cold!!See step 12 Step 17: Centrifuge precipitate to a pellet Step 18-21: Save supernatant (1ml in microfuge tube). Suspend pellets in 5ml cold buffer Step 22, 23: Add PMSF and place suspended pellet in dialysis tubing and give to TA

37. Today group 2 Set up standard assay as on page 142 –Measure loss of absorbance as NADH is converted to NAD+ Step 4 is similar to Kinetic curve you did for ADH (page 124) only reversed as measure loss of absorbance Steps 8-12: You will determine the velocity of LDH catalyzed reaction by varying the concentration of LDH with constant substrate and cofactor. Be sure to adjust the amount of reaction buffer to give 3.2 ml final volume in each assay

38. Very Important: Page 145 Blank without NADH Blank with NADH

39. Today group 2 continued You are establishing the assay conditions you will use next week to follow the purification of LDH. You must become proficient at this assay.

40. Flow chart 1B (page 142)

41. Spurious Vo Measurements Same as with ADH (this is similar to your [ADH] exp)

42. Procedure (Page 143) 1 Step 1-6. Will create a kinetic curve for LDH (adjust volume of buffer to make 3.2ml) –Similar to ADH 2. Repeat kinetic curve with different concentrations of enzyme –This is protocol you will use as you purify LDH Do this assay on the unknown samples from step one and 2a from group 1.

43. C2-3. Page 144 Table C.2-3. Lactate Dehydrogenase Reaction Time Courses Reading number time (seconds) A 340 readings 50 µl sample 100 µl sample 200 µl sample 300 µl sample 400 µl sample 10 215 330 445 560 675 790 8105 9120

44. Today 200 microliter

45. Next Week Column Chromatography Due next time: Prelab assignment for period 2 of ‘LDH Purification’ You really should write up or otherwise arrange what you did today as soon as possible. Do Not Trust Your Memory

46. Next lab Need member of group to be here at 1:30 to begin washing column Will need to measure absorbance at 280 to determine that contaminating protein is lost from column. Wash and measure until A 280 is constant.

47. Strategy For samples generated determine amount of protein (A 280 ) and activity Activity per microgram of protein =s specific activity You strive for maximal activity per unit of protein. (table C2-4 Column G, Page 162)

48. Will generate this elution profile Page 153

49. Will fill out this critical table as we proceed page 162 (day 4) Table C.2-4. Enzyme Purification Table Net volume (ml) V 0 units per ml V 0 units Total (an “amount”) Protein content (% of total) Protein concentration (mg/ml) Net amount of protein (mg) Specific Activity (V 0 /mg protein) StepABCDEFG 1. Cleared 2. (NH 4 ) 2 SO 4 Supernata nt 3. diluted dialyzed sample/ solution placed on column 4. pooled peak tubes from column Column C = (Column A)(Column B) Column F = (Column A)(Column E) Column G = Column C/Column F = Column B / Column E Column D = Column C/first value in Column C