Background: As the newest and least experienced student in a biochemistry research lab, you spend your first few weeks washing glassware and labeling test tubes. You then graduate to making buffers and stock solutions for use in various laboratory procedures. Finally you are given responsibility for purifying a protein. It is a citric acid cycle (Kreb’s cycle) enzyme, citrate synthase, located in the mitochondrial matrix. Following an established protocol previously published by your lab, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply an explanation of the purpose and intended outcome of each step.
You may use both text and online resources. Some useful references might be:
Lehninger Principles of Biochemistry, 4th ed. Nelson & Cox (Freeman, 2005)
Biochemistry, 7 ed. Berg, Tymczenco & Stryer (Freeman, 2012)
Protein Purification: Principles and Practice, 3rd ed. Scopes (Springer-Verlag, 1994)
Experimental Biochemistry, 3rd ed. Switzer & Garrity (Freeman, 1999)
Modern Experimental Biochemistry, 3rd ed. Boyer (Benjamin Cummings, 2000)
Experiments in Biochemistry, 2nd ed. Farrell & Taylor (Thomson/Brooks Cole, 2005)
Biochemistry Concepts and Connections, 1st ed. Appling (Pearson)
Objectives: after this experiment, you will be able to
- Understand the principles of fractionating a tissue homogenate
- Predict the solubility of proteins in solutions of various salt concentrations (selective precipitation)
- Clarify the use of centrifugation in purification of a specific component
- Discuss the basis of dialysis and how it partially separates a mixture of solutes
- Explain the purpose and apply the principles of several types of chromatography
- Understand electrophoresis and isoelectric focusing
Reference texts and an online connection
The following questions need to be answered in Canvas. You will write an explanation for each step in the protocol (guiding questions may suggest some factors to address) and state its intended outcome. Plagiarized answers will receive no credit.
Part A. You pick up 20 kg of beef hearts from a nearby meat market. You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef hearts in a high-speed blender in several liters of a medium containing 0.2 M sucrose (rather than in plain water) buffered to pH 7.2.
Why do you use beef heart tissue and in such a large quantity? What is the purpose of keeping it cold? What happens to the tissue when it is homogenized? Why suspend the homogenate in 0.2 M sucrose rather than water and why add buffer to maintain pH 7.2?
Part B. You subject the resulting heart homogenate, which is dense and opaque, to a series of centrifugations of increasing centrifuge speed and for longer times. You discard the pellet and retain the supernatant each time.
What does this accomplish? What is in the discarded pellets?
Part C. You retain the supernatant fraction that contains mostly intact mitochondria and work with it further. Next you osmotically lyse (burst open) the mitochondria. The lysate, which is less dense than the homogenate but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate, you add ammonium sulfate, a highly soluble salt, to a specific concentration. You centrifuge the solution, decant and save the supernatant and discard the pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample but this time you save the pellet because it contains the protein of interest.
What is the rationale behind the two-step addition of salt? Is the pellet a pure sample of the protein of interest?
Part D. You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze the sample overnight in a large volume of buffered (pH 7.2) solution, which is exchanged several times with fresh solution.
Why isn’t ammonium sulfate part of the dialysis solution? What happens to the salt concentration inside the dialysis membrane and in the soaking solution? What happens to the mitochondrial proteins by the end of this dialysis step?
Part E. You run the dialyzed solution through a size-exclusion gel chromatographic column. You collect the first fraction to elute off the column that has appreciable protein concentration, as detected by measuring the UV absorbance at 280 nm.
What does the instruction to “collect the first fraction off the column” tell you about the protein we’re trying to purify? Why is UV absorbance at 280 nm a good way to monitor for the presence of proteins in the eluted fractions?
Part F. You place the chromatography fraction collected in e) on a cation-exchange chromatographic column. After discarding the initial solution that elutes off the column (the flowthrough), you add a “washing solution” of higher pH to the column and collect the protein fraction that immediately elutes.
Explain what you are doing. What changes in your protein when the initial pH 7.2 solution is replaced by one with higher pH?
Part G. You run a small sample of your solution, now greatly reduced in volume and quite clear, on an isoelectric focusing gel. When stained, the gel shows three sharp bands. The purification protocol you are following says that the protein of interest is in the band with pI of 5.6. You cut out the pI = 5.6 band from the isoelectric focusing gel, solubilize the protein(s) and subject the sample to SDS polyacrylamide gel electrophoresis (SDS-PAGE). The protein resolves as a single band.
Explain how the isoelectric focusing and SDS polyacrylamide gel electrophoresis techniques work. Is it certain that the pI 5.6 band on the isoelectric focusing gel was a single pure protein? What do the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing?
Part H. You redissolve your purified enzyme in buffered solvent, determine its activity in a standard assay procedure then proceed to investigate its properties.
What (order of magnitude) yield of pure enzyme might you expect to recover from your 20 kg sample of beef hearts if you work with reasonable efficiency at each step? How would the total mass of protein and the specific activity of your sample change over the course of the purification procedure?