Protein Purification and Characterization Techniques

Purification of the Enzyme Xanthine Dehydrogenase from a Fungus 

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Many different proteins exist in a single cell. A detailed study of the properties of any one protein requires a homogeneous sample consisting of only one kind of molecule. The separation and isolation, or purification, of proteins constitutes an essential first step to further experimentation. In general, separation techniques focus on size, charge, and polarity—the sources of differences between molecules. Many techniques are performed to eliminate contaminants and to arrive at a pure sample of the protein of interest. As the purification steps are followed, we make a table of the recovery and purity of the protein to gauge our success. Table 5.1 shows a typical purification for an enzyme. The percent recovery column tracks how much of the protein of interest has been retained at each step. This number usually drops steadily during the purification, and we hope that by the time the protein is pure, sufficient product will be left for study and characterization. The specific activity column compares the purity of the protein at each step, and this value should go up if the purification is successful. 

Column chromatography is widely used to purify proteins. 

To begin the process of purification, proteins are released from cells with homogenization using a variety of physical techniques. 

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Methods for Purification and Characterization of Proteins, the primary goal is to acquaint the reader with the techniques used for protein purification. The first part of chapter 6 presents methods for protein fractionation. In the second part of this chapter, purification procedures for two proteins, UMP synthase and lactose carrier protein, are presented so that the student can see how different purification steps are combined for maximum effectiveness. 

This chapter begins with an introduction of general considerations for protein purification and then focuses on purification strategies and characterization technique for MCOs. Examples of MCO purification and characterization will regularly be referenced and the chapter will pay particular attention to the characterization of metals centers, as expression, purification, and characterization of copper-dependent MCOs require some unique methods. Techniques to characterize MCO activity and behavior after immobilization onto electrode surfaces will not be covered, as these techniques are described elsewhere in the book. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

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