2 Структура и функция белка (1160071), страница 10
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Enzymes are usually assayed at their optimum pH and atsome convenient temperature within the range 25 to 38 °C. Also, veryhigh substrate concentrations are generally required so that the initialreaction rate, which is measured experimentally, is proportional toenzyme concentration (Chapter 8).By international agreement, 1.0 unit of enzyme activity is definedas the amount of enzyme causing transformation of 1.0 fimol of substrate per minute at 25 °C under optimal conditions of measurement.The term activity refers to the total units of enzyme in the solution.The specific activity is the number of enzyme units per milligram ofprotein (Fig. 6-3).
The specific activity is a measure of enzyme purity:it increases during purification of an enzyme and becomes maximaland constant when the enzyme is pure (Table 6-4).After each purification step, the activity of the preparation (inunits) is assayed, the total amount of protein is determined independently, and their ratio gives the specific activity. Activity and totalprotein generally decrease with each step. Activity decreases becausesome loss always occurs due to inactivation or nonideal interactionswith chromatographic materials or other molecules in the solution.Total protein decreases because the objective is to remove as muchnonspecific protein as possible.
In a successful step, the loss of nonspecific protein is much greater than the loss of activity; therefore, specificactivity increases even as total activity falls. The data are then assembled in a purification table (Table 6-4).
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 (by methodsto be described later).For proteins that are not enzymes, other quantification methodsare required. Transport proteins can be assayed by their binding to themolecule they transport, and hormones and toxins by the biologicaleffect they produce; for example, growth hormones will stimulate thegrowth of certain cultured cells. Some structural proteins representsuch a large fraction of a tissue mass that they can be readily extractedand purified without an assay. The approaches are as varied as theproteins themselves.Figure 6-3 Activity versus specific activity.
Thedifference between these two terms can be illustrated by considering two jars of marbles. The jarscontain the same number of red marbles (representing an unknown protein), but different amountsof marbles of other colors. If the marbles are takento represent proteins, both jars contain the sameactivity of the protein represented by the red marbles.
The second jar, however, has the higher specific activity because here the red marbles represent a much higher fraction of the total.Chapter 6 An Introduction to Proteinsuuu&uuuuuu141Directionofmigration(a)Proteins Can Be Characterized by ElectrophoresisIn addition to chromatography, another important set of methods isavailable for the separation of proteins, based on the migration ofcharged proteins in an electric field, a process called electrophoresis.These procedures are not often used to purify proteins in largeamounts because simpler alternative methods are usually availableand electrophoretic methods often inactivate proteins.
Electrophoresisis, however, especially useful as an analytical method. Its advantage isthat proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or thedegree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as itsisoelectric point and approximate molecular weight.In electrophoresis, the force moving the macromolecule (nucleicacids as well as proteins are separated this way) is the electrical potential, E.
The electrophoretic mobility of the molecule, /x, is the ratio ofthe velocity of the particle, V, to the electrical potential. Electrophoretic mobility is also equal to the net charge of the molecule, Z, dividedby the frictional coefficient, f. Thus:VZElectrophoresis of proteins is generally carried out in gels made upof the cross-linked polymer polyacrylamide (Fig. 6-4). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteinsapproximately in proportion to their mass, or molecular weight.An electrophoretic method commonly used for estimation of purityand molecular weight makes use of the detergent sodium dodecylsulfate (SDS).
SDS binds to most proteins (probably by hydrophobicinteractions; see Chapter 4) in amounts roughly proportional to themolecular weight of the protein, about one molecule of SDS for everytwo amino acid residues. The bound SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant.(b)Figure 6-4 Electrophoresis. (a) Different samplesare loaded in wells or depressions at the top of thepolyacrylamide gel. The proteins move into the gelwhen an electric field is applied. The gel minimizesconvection currents caused by small temperaturegradients, and it minimizes protein movementsother than those induced by the electric field,(b) Proteins can be visualized after electrophoresisby treating the gel with a stain such as Coomassieblue, which binds to the proteins but not to the gelitself.
Each band on the gel represents a differentprotein (or protein subunit); smaller proteins arefound nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase from the bacterium E. coli. The first lane showsthe proteins present in the crude cellular extract.Successive lanes show the proteins present aftereach purification step. The purified protein containsfour subunits, as seen in the last lane on the right.ONa+II"0—S—O—(CH 2 ) 11 CH 3OSodium dodecyl sulfate(SDS)142Part II Structure and CatalysisIn addition, the native conformation of a protein is altered when SDS isbound, and most proteins assume a similar shape, and thus a similarratio of charge to mass. Electrophoresis in the presence of SDS therefore separates proteins almost exclusively on the basis of mass (molecular weight), with smaller polypeptides migrating more rapidly.
Afterelectrophoresis, the proteins are visualized by adding a dye such asCoomassie blue (Fig. 6-4b) which binds to proteins but not to the gelitself. This type of gel provides one method to monitor progress in isolating a protein, because the number of protein bands should decreaseas the purification proceeds. When compared with the positions towhich proteins of known molecular weight migrate in the gel, the position of an unknown protein can provide an excellent measure of itsmolecular weight (Fig. 6-5).
If the protein has two or more differentsubunits, each subunit will generally be separated by the SDS treatment, and a separate band will appear for each.Figure 6—5 Estimating the molecular weight of aprotein. The electrophoretic mobility of a protein onan SDS polyacrylamide gel is related to its molecular weight, Mr.
(a) Standard proteins of knownmolecular weight are subjected to electrophoresis(lane 1). These marker proteins can be used to estimate the Mr of an unknown protein (lane 2). (b) Aplot of log MT of the marker proteins versus relative migration during electrophoresis allows the Mrof the unknown protein to be read from the graph.0Myosin 200,000(3-Galactosidase 116,250Phosphorylase b 97,400Bovine serum albumin66,200Ovalbumin45,000Carbonic anhydrase31,000Soybean trypsin inhibitorLysozyme21,50014,400©Table 6-5 The isoelectric points of someproteinspiPepsinEgg albuminSerum albuminUrease/3-LactoglobulinHemoglobinMyoglobinChymotrypsinogenCytochrome cLysozyme-1.04.64.95.05.26.87.09.510.711.0MYUnknownstandards proteinRelative migration(a)(b)Isoelectric focusing is a procedure used to determine the isoelectric point (pi) of a protein (Fig.
6-6). A pH gradient is established byallowing a mixture of low molecular weight organic acids and bases(ampholytes; see p. 118) to distribute themselves in an electric fieldgenerated across the gel. When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pi. Proteins withdifferent isoelectric points are thus distributed differently throughoutthe gel (Table 6-5).Combining these two electrophoretic methods in two-dimensionalgels permits the resolution of complex mixtures of proteins (Fig.
6-7).This is a more sensitive analytical method than either isoelectric focusing or SDS electrophoresis alone. Two-dimensional electrophoresisseparates proteins of identical molecular weight that differ in pi, orproteins with similar pi values but different molecular weights.143Isoelectric focusingDecreasingpiFirstdimensionAn ampholytesolution ispH9incorporatedinto a gel.Seconddimension'I©i©A stable pHgradient isestablished inthe gel afterapplication ofan electricfield.Proteinsolution isadded andelectric fieldis reapplied.Afterstaining,proteins areshown to bedistributedalong pHgradient.TwodimensionalJSDS polyacrylamidegel electrophoresisFigure 6-6 Isoelectric focusing.
This techniqueseparates proteins according to their isoelectricpoints. A stable pH gradient is established in thegel by the addition of appropriate ampholytes. Aprotein mixture is placed in a well on the gel. Withan applied electric field, proteins enter the gel andmigrate until each reaches a pH equivalent to itspi. Remember that the net charge of a protein iszero when pH = pi.^^Decreasingr1Decreasing ,pi(a)it "I- --*Figure 6—7 Two-dimensional electrophoresis.(a) Proteins are first separated by isoelectric focusing.
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