Human Biochemistry Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Enzymes: General Properties
Enzyme Purification

All information regarding individual metabolic reactions, intermediates generated at successive stages of Various metabolic pathways, and The regulatory mechanisms of catalysts has been obtained primarily through The Use of purified enzyme preparations. Highly purified enzyme preparations are also essential for acquiring reliable data on enzyme kinetics, Cofactors, active sites, Structure, and MECHANISM OF ACTION.

The purification process involves isolating the enzyme of interest from a crude Cell extract containing a multitude of other components. Small molecules are removed by dialysis or Gel filtration; Nucleic Acids are eliminated via precipitation by adding the antibiotic streptomycin, and so on. The primary challenge is to separate the target enzyme from hundreds of chemically and physically similar Proteins.

Classical Purification Methods

The following purification methods are widely used: precipitation at varying salt concentrations (most commonly ammonium sulfate or sodium sulfate) or with organic Solvents (acetone, ethanol); differential Denaturation via heating or pH adjustment; differential centrifugation, gel filtration, and Electrophoresis.

For large-scale and rapid Protein Purification, selective Adsorption and Elution using the Cellulose anion exchanger diethylaminoethylcellulose (DEAE-cellulose) and the cation exchanger carboxymethylcellulose are successfully employed. Size-based protein Separation using molecular sieves, such as Sephadex, is also widespread. However, these methods are relatively low-selectivity (unless used in combination) when it comes to isolating an individual protein from a complex mixture. This challenge is more readily addressed by Affinity Chromatography.

A typical Procedure for purifying a Liver enzyme with a high yield and a 490-fold purification factor is outlined in Table 7.2. Note the changes in specific activity and enzyme yield during the purification process. The procedure is designed to achieve maximum specific activity (units of enzyme activity per milligram of protein) while maintaining the highest possible recovery of the initial total activity.

Class="center">Table 7.2. Typical enzyme purification procedure

Affinity Chromatography

A remarkable advantage of this purification method is its ability to selectively isolate a single specific protein, or at least a small subset of proteins, from a complex mixture. The technique relies on an immobilized Ligand that specifically interacts with the target protein to be purified. Among all proteins present in the mixture, only those capable of strong interaction with the immobilized ligand will bind to it. After washing away all unbound proteins, the desired enzyme is eluted from the immobilized ligand using either concentrated salt solutions or a solution containing the soluble form of the ligand. Successful application of affinity chromatography yields striking purification results that typically surpass those achieved by the consecutive use of multiple classical methods.

Because Enzymes generally exhibit high Specificity for their substrates and cofactors, the most suitable ligands are substrate and cofactor derivatives covalently bound to a matrix, such as Sephadex. They can be attached to the matrix either directly or via a 3–8 carbon atom spacer arm (linker). Using a linker helps overcome steric hindrance issues where direct attachment of the ligand to the matrix might impede its interaction with the enzyme. However, the Introduction of a hydrophobic linker can occasionally complicate isolation due to hydrophobic ligand chromatography effects (see below). A successful application of affinity chromatography is the purification of numerous dehydrogenases using affinity matrices with NAD⁺ as the ligand. In such cases, multiple dehydrogenases may bind to the ligand and co-elute when treated with a NAD⁺ solution; their subsequent separation is then achieved using substrate affinity matrices rather than coenzyme ones, or by elution with "dead-end ternary complexes" containing the coenzyme, a specific substrate, and a specific product.

Affinity chromatography shares many similarities with dye-ligand chromatography (using Blue, Green, or Red Sepharose) and Hydrophobic Interaction Chromatography, where octyl- or phenyl-Sepharose serves as the matrix. In the former approach, an organic dye acting as an analog of a substrate, cofactor, or allosteric effector is employed as the immobilized ligand. Elution is typically carried out using a salt gradient of increasing concentration.

In hydrophobic interaction chromatography, alkyl or aryl Hydrocarbons are attached to a matrix (such as Sephadex). Protein binding to these Supports is driven by hydrophobic interactions between the alkyl chains and hydrophobic patches on the protein molecule. Proteins are loaded in solutions with high salt concentrations, such as (NH₄)₂SO₄, and eluted using a decreasing salt gradient.

Assessing the Homogeneity of Protein Preparations by Polyacrylamide gel electrophoresis

The homogeneity of protein preparations is best evaluated using polyacrylamide gel electrophoresis under various conditions. One-dimensional electrophoresis of the native protein (provided sufficient sample is available) can reveal both Major and minor protein impurities. In the two-dimensional variant (O'Farrell's method), denatured proteins are separated in the first dimension according to their pI values (in the presence of urea) along a pH gradient generated by dimerized ampholytes. In the second dimension, proteins denatured with sodium dodecyl sulfate are separated based on the molecular weights of their protomers (if the protein is oligomeric).