Objectives

Introduction

Many types of salts have been employed to effect protein separation and purification through salting-out. Of these salts, ammonium sulfate has been the most widely used chemical because it has high solubility and is relatively inexpensive. Because enzymes are proteins, enzyme purification can be carried out by following the same set of procedures as those for protein, except that some attention must be paid to the consideration of permanent loss of activity due to denaturation under adverse conditions.

There are two major salting-out procedures. In the first procedure, either a saturated salt solution or powdered salt crystals are slowly added to the protein mixture to bring up the salt concentration of the mixture. For example, the salt concentration reaches 25% saturation when 1 ml of the saturated salt solution is added to 3 ml of the salt-free protein solution; 50% for 3 ml added; 75% for 9 ml added; and so on. The precipitated protein is collected and categorized according to the concentration of the salt solution at which it is formed. This partial collection of the separated product is called fractionation. For example, the fraction of the precipitated protein collected between 20 and 21% of salt saturation is commonly referred to as the 20-21% fraction. The protein fractions collected during the earlier stages of salt addition are less soluble in the salt solution than the fractions collected later.

Whereas the first method just described uses increasing salt concentrations, the following alternative method uses decreasing salt concentrations. In this alternative method, as much protein as possible is first precipitated with a concentrated salt solution. Then a series of cold (near 0ºC) ammonium sulfate solutions of decreasing concentrations are employed to extract selectively the protein components that are the most soluble at higher ammonium sulfate concentrations. The extracted protein is recrystallized and thus recovered by gradually warming the the cold solution to room temperature. This method has the added advantages that the extraction media may be buffered or stabilizing agents be added to retain the maximum enzyme activity. The efficiency of recovery typically ranges from 30 to 90%, depending on the protein. The recrystallization of protein upon transferring the extract to room temperature may occur immediately or may sometimes take many hours. Nevertheless, very rarely does recrystallization fail to occur. The presence of fine crystals in a solution can be visually detected from the turbidity.

List of Reagents and Instruments

A. Equipment

• Test tubes
• Graduated cylinder
• Pipets
• Balance
• Centrifuge
• Filtration devices

B. Reagents

• Protein solution, 1.0 g/l hemoglobin, 200 ml
• (May need centrifugation to obtain a clear solution.)
• Fungal alpha-amylase, 10 g/l
• Protease, 10 g/l
• Saturated (NH₄)₂SO₄ solution. See Note 1.

Procedures

1. Isolation of Hemoglobin:
   • Record the absorbance of the hemoglobin solution. Suggested wavelength: 577nm. This measurement is to be used in the calculation of the recovery of the protein.
   • Pipet 4 ml of the hemoglobin solution into a test tube.
   • While stirring, add the saturated ammonium sulfate solution drop-wise to the protein solution until precipitates start to form. In order to record accurately the amount of ammonium sulfate solution added, the salt solution should be dispensed from a graduated pipet or a buret. It is critical to avoid the spatial nonuniformity in the salt concentration during the addition of the salt solution. Localized concentration hot spots will prematurely initiate the precipitation of other proteins and inadvertently affect the purify of the protein crystals. Record the volume of the saturated ammonium sulfate solution needed to cause precipitation. Also note that protein precipitation is not instantaneous; it may require 15--20 minutes to equilibrate.
   • Centrifuge the mixture at 10,000 g for 15 minutes. Collect the precipitate by carefully discarding as much supernatant as possible.
   • Reconstitute the original hemoglobin solution by resuspending the precipitate in 4 ml of water. This can be done by first adding approximately 2 ml of water from a water bottle to the centrifuge tube, shaking the test tube to redissolve the precipitate, and transferring as much as possible the hemoglobin solution in the centrifuge tube into a test tube with a pipet while noting the volume. Rinse the centrifuge tube with another ml of water, pipetting this rinse in the test tube as well, again, while noting the volume transferred. Finally, add the residual water to bring the total volume in the test tube to 4 ml.
   • Measure the absorbance of the reconstituted hemoglobin solution with a spectrophotometer.
2. Isolation of Fungal alpha-Amylase:
   • Instead of hemoglobin solution, use 4ml of 20 g/l of fungal alpha-amylase.
   • Salt-out the enzyme with a saturated ammonium sulfate solution as in Step 1. Record the volume of the saturated salt solution added. Collect the protein precipitates. The precipitates may be collected by following a similar washing procedure as in Step 1. Try not to dilute the enzyme solution too much. Filtration through a syringe filter unit may be conveniently employed if the crystals are not too small and if the collected crystals can be easily washed off the filter paper. Usually the quantitative analysis of the activity of the enzyme collected with the filtration method is not as accurate as the centrifugation method.
   • After resuspending the proteins, analyze the enzyme activity by the methods introduced in the previous experiments.
3. Isolation of Protease:
   • Same as Step 2, except that 4 ml of saturated protease is used. Note that protease may not be totally soluble, and supernatant can be obtained by centrifugation.
4. Isolation of a mixture of Hemoglobin, alpha-Amylase, and Protease:
   • Pursue this step if the student believes that at least one of the enzymes, alpha-amylase or protease, can be effectively separated by the ammonium sulfate salting-out procedure based on the results of Steps 1-3.
   • Add 1ml of hemoglobin solution, 4 ml of 10g/l of alpha-amylase, and 4 ml of 10g/l protease into a test tube.
   • Collect the protein precipitate fractions as they are formed. If there are more components to be separated from the supernatant, pour out the supernatant and subject it to further treatment with the saturated salt solution. Otherwise, discard the supernatant.
   • Analyze the enzyme activities of each fraction on starch and protein solutions.

Notes

1. Add 750 g of ammonium sulfate to 1000 ml of water in a beaker or flask. Simply stir the solution at room temperature with a magnetic stirrer for 15 minutes or until saturation. Gently decant the clear supernatant solution after the undissolved solids settle on the bottom of the flask. (Filtration is not really necessary.)

Discussions

The recovery of protein can have very significant economical implications. Because a fixed fraction of the original protein stays soluble in the solution, the recovery of protein is often not near 100%. Of course, a yield of over 100% indicates that there may be problems associated with the assay method.

In a typical protein preparation or purification step carried out in a laboratory where the aim is to isolate a small quantity of a product for structural or kinetic studies, a saturated ammonium sulfate solution is routinely used. It is also the procedure taken in this experiment. However, in an actual large scale commercial process, it is better to add ammonium sulfate directly into the protein mixture as powdered solids so that the effect of dilution by the salt solution is minimized.

Questions

1. From the volume of the saturated ammonium sulfate added to the protein solution at the onset of precipitation, calculate the salt concentration in terms of percent saturation. Note that this is the same as a dilution calculation. Did the supernatant have the characteristic red/brown color of hemoglobin? (Oxygenated hemoglobin is red, and the deoxygenated form is brown.) What does the color in the supernatant, if any, indicate? From the absorbance readings before and after the protein separation, how much many percent of the original protein is recovered in the ammonium sulfate salting-out procedure?
2. At what salt concentration did alpha-amylase precipitate? Was there any enzyme denaturation? If yes, suggest an alternate procedure of isolation. Answer the same set of questions for protease.
3. Did you think alpha-amylase and protease could be separated from each other and from hemoglobin? Why? If enzyme separation from a mixture was attempted, were you successful in separating the mixture into respective components? If yes, what fraction was alpha-amylase? What fraction was protease? Was there any shift in the incipient precipitation concentrations of the salt solution? Was there any decrease in the enzyme activities?
4. Comment on ways to improve the experiment.

References

1. Jakoby, W.B., Crystallization as a purification technique, Enzyme Purification and Related Techniques, in Methods in Enzymology, Vol. 22, Jakoby, W.B., Ed., Academic Press, 1971.