CH339K

1. CH339K Physical Methods: How to Purify and Sequence a Weapons-Grade Protein

2. First Question How do I measure the amount of protein I have?

3. UV Absorption Spectrophotometry

5. Second Question How can I spot my protein in the great mass of different proteins?

6. Electrophoresis

8. The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so: i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein

9. Polyacrylamide Gels

11. Polyacrylamide gel electrophoresis of whole cell proteins of three strains of lactic acid bacteria.

12. Agarose Gelidium sp.

14. SDS PAGE Sodium Dodecyl (Lauryl) Sulfate SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein

15. Constant q/M ratio

18. Disulfide cleavage

19. Disulfide cleavage and chain separation + bME

20. Isoelectric Point

22. Isoelectric Focusing

23. pH

24. Carrier Ampholytes • Amphoteric Electrolytes • Mixture of molecules containing multiple amino- and carboxyl- groups with closely spaced pIs • Partition into a smooth, buffered pH gradient

25. Separation by pI

26. Isoelectric Focusing Below the pI, a protein has a positive charge and migrates toward the cathode Above the pI, a protein has a negative charge and migrates toward the anode

27. Isoelectric FocusingFoot Flesh Extracts from Pomacea flagellata and Pomacea patula catemacensis

28. Protein Purification Steps 1 unit = amount of enzyme that catalyzes conversion of 1 mmol of substrate to product in 1 minute

29. Purification visualized

30. Example:Purification of Ricin

31. Georgi Markov 1929-1978

33. Ricinus communis – castor oil plant

34. Ricin Ricin B chain (the attachment bit)

35. Ricin Action • Ricin and related enzymes remove an adenine base from the large ribosomal RNA • Shut down protein synthesis

36. The possibility that ricin might be used as an asymmetric warfare weapon has not escaped the attention of the armed services. The last time I was qualified to know for sure, there were no effective antidotes.

37. Raw Extract (NH4)2SO4 Cut Affinity Gel Filtration

38. Salting In – Salting out • salting in: Increasing ionic strength increases protein solubility • salting out: Increasing further leads to a loss of solubility

39. Salting in – salting out The solubility of haemoglobin in different electrolytes as a function of ionic strength. Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47

40. Salting in: Counterions help prevent formation of interchain salt links Solubility reaches minimum at pI

41. Salting out: there’s simply less water available to solubilize the protein.

42. Different proteins have different solubilities in (NH4)2SO4

43. Lyotropic  ChaotropicSeries Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+>Al3+> guanidinium / urea Anions: SO42−> HPO42−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3−> ClO4−> SCN−

44. Bring to 37% Saturation – ricin still soluble, many other proteins ppt • Collect supernatant • Bring to 67% Saturation – ricin ppt, many remaining proteins still soluble • Collect pellet • Redissolve in buffer

45. Dialysis and Ultrafiltration(How do you get the %@$&#! salt out?)

46. Raw Extract (NH4)2SO4 Cut Affinity Gel Filtration

47. Separation by chromatography Basic Idea: You have a stationary phase You have a mobile phase Your material partitions out between the phases.

48. Affinity Chromatography

50. Structure of Agarose Agarose is a polymer of agarobiose, which in turn consists of one unit each of galactose and 3,6-anhydro-a-L-galactose. Ricin sticks to galactose, so store-bought agarose acts as an affinity column right out of the bottle, with ricin binding the beads while other proteins wash through.