Working with Proteins: -Unseparated Proteins Can Be Quantified

To purify a protein, it is essential to have a way of detecting and quantifying that protein in the presence of many other proteins at each stage of the procedure. Often, purification must proceed in the absence of any information about the size and physical properties of the protein or about the fraction of the total protein mass it represents in the extract. For proteins that are en zymes, the amount in a given solution or tissue extract can be measured, or assayed, in terms of the catalytic effect the enzyme produces—that is, the increase in the rate at which its substrate is converted to reaction products when the enzyme is present. For this purpose one must know (1) the overall equation of the reaction catalyzed, (2) an analytical procedure for determining the disappearance of the substrate or the appearance of a reaction product, (3) whether the enzyme requires co factors such as metal ions or coenzymes, (4) the dependence of the enzyme activity on substrate concentration, (5) the optimum pH, and (6) a temperature zone in which the enzyme is stable and has high activity. Enzymes are usually assayed at their optimum pH and at some convenient temperature within the range 25 to 38 C. Also, very high substrate concentrations are generally used so that the initial reaction rate, measured experimentally, is proportional to enzyme concentration (Chapter 6). By international agreement, 1.0 unit of enzyme ac tivity is defined as the amount of enzyme causing trans formation of 1.0 mol of substrate per minute at 25 C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3–23). The specific activity is a measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3–5).

FIGURE 3–22 Two-dimensional electrophoresis. (a) Proteins are first separated by isoelectric focusing in a cylindrical gel. The gel is then laid horizontally on a second, slab-shaped gel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. Horizontal separation reflects differences in pI; vertical separation reflects differences in molecular weight. (b) More than 1,000 different proteins from E. coli can be resolved using this technique.

After each purification step, the activity of the preparation (in units of enzyme activity) is assayed, the total amount of protein is determined independently, and the ratio of the two gives the specific activity. Ac tivity and total protein generally decrease with each step. Activity decreases because some loss always oc curs due to inactivation or nonideal interactions with chromatographic materials or other molecules in the solution. Total protein decreases because the objective is to remove as much unwanted or nonspecific protein as possible. In a successful step, the loss of nonspecific protein is much greater than the loss of activity; therefore, specific activity increases even as total activity falls. The data are then assembled in a purification table similar to Table 3–5. A protein is generally considered pure when further purification steps fail to increase specific activity and when only a single protein species can be detected (for example, by electrophoresis). For proteins that are not enzymes, other quantification methods are required. Transport proteins can be assayed by their binding to the molecule they transport, and hormones and toxins by the biological effect they produce; for example, growth hormones will stimulate the growth of certain cultured cells. Some structural proteins represent such a large fraction of a tissue mass that they can be readily extracted and purified without a functional assay. The approaches are as varied as the proteins themselves.

FIGURE 3–23 Activity versus specific activity. The difference between these two terms can be illustrated by considering two beakers of marbles. The beakers contain the same number of red marbles, but different numbers of marbles of other colors. If the marbles represent proteins, both beakers contain the same activity of the protein represented by the red marbles. The second beaker, however, has the higher specific activity because here the red marbles represent a much higher fraction of the total.

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مواضيع ذات صلة

The Covalent Structure of Proteins:-Large Proteins Must Be Sequenced in Smaller Segments

The Covalent Structure of Proteins:- Short Polypeptides Are Sequenced Using Automated Procedures

The Covalent Structure of Proteins:-The Amino Acid Sequences of Millions of Proteins Have Been Determined

The Covalent Structure of Proteins:-The Function of a Protein Depends on Its Amino Acid Sequence

Working with Proteins:- Proteins Can Be Separated and Characterized by Electrophoresis

Working with Proteins:- Proteins Can Be Separated and Purified

Peptides and Proteins:- Some Proteins Contain Chemical Groups Other Than Amino Acids

Peptides and Proteins: - Polypeptides Have Characteristic Amino Acid Compositions

Peptides and Proteins: -Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes

Peptides and Proteins: -Peptides Can Be Distinguished by Their Ionization Behavior

Peptides and Proteins: - Peptides Are Chains of Amino Acids

Amino Acids: -Amino Acids Differ in Their Acid-Base Properties