2 Структура и функция белка (1160071), страница 11
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The gel is then laid horizontally on a secondgel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. In this two-dimensional gel, horizontal separation reflects differencesin pi; vertical separation reflects differences inmolecular weight, (b) More than 1,000 differentproteins from E. coli can be resolved using thistechnique.(b)iPart II Structure and Catalysis144The Antibody-Antigen Interaction Is Used toQuantify and Localize Proteins(a)Binding sitesAntibodyAntigen(b)Several sensitive analytical procedures have been developed from thestudy of a class of proteins called antibodies or immunoglobulins.Antibody molecules appear in the blood serum and certain tissues of avertebrate animal in response to injection of an antigen, a protein orother macromolecule foreign to that individual.
Each foreign proteinelicits the formation of a set of different antibodies, which can combinewith the antigen to form an antigen-antibody complex. The productionof antibodies is part of a general defense mechanism in vertebratescalled the immune response.Antibodies are Y-shaped proteins consisting of four polypeptidechains. They have two binding sites that are complementary to specificstructural features of the antigen molecule, making possible the formation of a three-dimensional lattice of alternating antigen and antibody molecules (Fig.
6-8). If sufficient antigen is present in a sample,the addition of antibodies or blood serum from an immunized animalwill result in the formation of a quantifiable precipitate. No such precipitate is formed when serum of an unimmunized animal is mixedwith the antigen.Antibodies are highly specific for the foreign proteins or other macromolecules that evoke their formation. It is this specificity that makesthem valuable analytical reagents. A rabbit antibody formed to horseserum albumin, for example, will combine with the latter but will notusually combine with other horse proteins, such as horse hemoglobin.Two types of antibody preparations are in use: polyclonal andmonoclonal.
Polyclonal antibodies are those produced by many different types (or populations) of antibody-producing cells in an animalimmunized with an antigen (in this case a protein). Each type of cellproduces an antibody that binds only to a specific, small part of theantigen protein. Consequently, polyclonal preparations contain a mixture of antibodies that recognize different parts of the protein. Monoclonal antibodies, in contrast, are synthesized by a population of identical cells (a clone) grown in cell culture. These antibodies arehomogeneous, all recognizing the same specific part of the protein.
Thetechniques for producing monoclonal antibodies were worked out byGeorges Kohler and Cesar Milstein.Antibodies are so exquisitely specific that they can in some casesdistinguish between two proteins differing by only a single amino acid.Figure 6-8 The immune response and the actionof antibodies, (a) A molecule of immunoglobulin G(IgG) consists of two polypeptides known as heavychains (white and light blue) and two known aslight chains (purple and dark blue). Immunoglobulins are glycoproteins and contain bound carbohydrate (yellow), (b) Each antigen evokes a specificset of antibodies, which will recognize and combineonly with that antigen or closely related molecules.(Antibody-binding sites are shown as red areas onthe antigen.) The Y-shaped antibodies each havetwo binding sites for the antigen, and can precipitate the antigen by forming an insoluble, latticelikeaggregate.Cesar MilsteinGeorges KohlerChapter 6 An Introduction to Proteins145fLJIStainedgelImmunoblot(b)Second antibody withlinked enzyme addedColor changeproportional toamount ofhormone present(a)When a mixture of proteins is added to a chromatography column inwhich the antibody is covalently attached to a resin, the antibody willspecifically bind its target protein and retain it on the column whileother proteins are washed through.
The target protein can then beeluted from the resin by a salt solution or some other agent. This canbe a powerful tool for protein purification.A variety of other analytical techniques rely on antibodies. In eachcase the antibody is attached to a radioactive label or some other reagent to make it easy to detect. The antibody binds the target protein,and the label reveals its presence in a solution or its location in a gel oreven a living cell. Several variations of this procedure are illustrated inFigure 6-9.
We shall examine some other aspects of antibodies inchapters to follow; they are of extreme importance in medicine and alsotell much about the structure of proteins and the action of genes.Figure 6-9 Analytical methods based on the interaction of antibodies with antigen, (a) An enzymelinked immunosorbent assay (ELISA) used in testing for human pregnancy. Human chorionic gonadotropin (hCG), a hormone produced by the placenta,is detectable in maternal urine a few days afterconception. In the ELISA, an antibody specific forhCG is attached to the bottom of a well in a plastictray, to which a few drops of urine are added. Ifany hCG is present, it will bind to the antibodies.The tube is then washed, and a second antibody(also specific for hCG) is added.
This second antibody is linked to an enzyme that catalyzes the conversion of a colorless compound to a colored one;the amount of colored compound produced providesa sensitive measure of the amount of hormone present. The ELISA has been adapted for use in determining the amount of specific proteins in tissuesamples, in blood, or in urine.(b) Immunoblot (or Western blot) technique.Proteins are separated by electrophoresis, thenantibodies are used to determine the presence andsize of the proteins. After separation, the proteinsare transferred electrophoretically from an SDS polyacrylamide gel to a special paper (which makesthem more accessible). Specific, labeled antibodyis added, then the paper is washed to remove unbound antibody. The label can be a radioactive element, a fluorescent compound, or an enzyme as inthe ELISA.
The position of the labeled antibodydefines the Mr of the detected protein. All of theproteins are seen in the stained gel; only theprotein bound to the antibody is seen in theimmunoblot.(c) In immunocytochemistry, labeled antibodiesare introduced into cells to reveal the subcellularlocation of a specific protein. Here, fluorescentlylabeled antibodies and a fluorescence microscopehave been used to locate tubulin filaments in ahuman fibroblast.146Part II Structure and CatalysisThe Covalent Structure of ProteinsAll proteins in all species, regardless of their function or biologicalactivity, are built from the same set of 20 amino acids (Chapter 5).What is it, then, that makes one protein an enzyme, another a hormone, another a structural protein, and still another an antibody? Howdo they differ chemically? Quite simply, proteins differ from each otherbecause each has a distinctive number and sequence of amino acid residues.
The amino acids are the alphabet of protein structure; they canbe arranged in an almost infinite number of sequences to make analmost infinite number of different proteins. A specific sequence ofamino acids folds up into a unique three-dimensional structure, andthis structure in turn determines the function of the protein.The amino acid sequence of a protein, or its primary structure,can be very informative to a biochemist. No other property so clearlydistinguishes one protein from another.
This now becomes the focus ofthe remainder of the chapter. We first consider empirical clues thatamino acid sequence and protein function are closely linked, then describe how amino acid sequence is determined, and finally outline themany uses to which this information can be put.The Function of a Protein Depends onIts Amino Acid SequenceThe bacterium E.
coli produces about 3,000 different proteins. Ahuman being produces 50,000 to 100,000 different proteins. In bothcases, each separate type of protein has a unique structure and thisstructure confers a unique function. Each separate type of protein alsohas a unique amino acid sequence. Intuition suggests that the aminoacid sequence must play a fundamental role in determining the threedimensional structure of the protein, and ultimately its function, but isthis expectation correct? A quick survey of proteins and how they varyin amino acid sequence provides a number of empirical clues that helpsubstantiate the important relationship between amino acid sequenceand biological function.
First, as we have already noted, proteins withdifferent functions always have different amino acid sequences. Second, more than 1,400 human genetic diseases have been traced to theproduction of defective proteins (Table 6-6). Perhaps a third of theseproteins are defective because of a single change in the amino acidsequence; hence, if the primary structure is altered, the function of theprotein may also be changed. Finally, on comparing proteins with similar functions from different species, we find that these proteins oftenhave similar amino acid sequences. An extreme case is ubiquitin, a 76amino acid protein involved in regulating the degradation of other proteins.
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