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This technique primarily exploits differences in the signand magnitude of the net electric charges of amino acids at a given pH,which are predictable from their pKa values or their titration curves.The chromatographic column consists of a long tube filled withparticles of a synthetic resin containing fixed charged groups; thosewith fixed anionic groups are called cation-exchange resins andthose with fixed cationic groups, anion-exchange resins. A simpleform of ion-exchange chromatography on a cation-exchange resin isdescribed in Figure 5-12. The affinity of each amino acid for the resinis affected by pH (which determines the ionization state of the molecule) and the concentration of other salt ions that may compete withthe resin by associating with the amino acid. Separation of amino acidscan therefore be optimized by gradually changing the pH and/or saltconcentration of the solution being passed through the column so as tocreate a pH or salt gradient.
A modern enhancement of this and otherchromatographic techniques is called high-performance liquidchromatography (HPLC). This takes advantage of stronger resinmaterial and improved apparatus designed to permit chromatographyat high pressures, allowing better separations in a much shorter time.For amino acids, the entire procedure has been automated, so thatelution, collection of fractions, analysis of each fraction, and recordingof data are performed automatically in an amino-acid analyzer. Figure 5-13 shows a chromatogram of an amino acid mixture analyzed inthis way.Amino Acids Undergo Characteristic Chemical ReactionsAs for all organic compounds, the chemical reactions of amino acids arethose characteristic of their functional groups.
Because all amino acidscontain amino and carboxyl groups, all will undergo chemical reactionscharacteristic for these groups. For example, their amino groups can beacetylated or formylated, and their carboxyl groups can be esterified.We will not examine all such organic reactions of amino acids, butseveral widely used reactions are noteworthy because they greatlysimplify the detection, measurement, and identification of amino acids.Chapter 5 Amino Acids and Peptides123Reservoir of buffer allows sample topercolate slowly through column.Solution ofamino acidsat pH 3.0is pouredonto acationexchangecolumn.Na~~O,S —,O*rjlR;11H,X - C - C O OH\m.0°QR1J-C-COOAmino acids with greatest positivecharge (red) bind the columnmost tightly and therefore movemost slowly.
Those with the leastamount of positive charge (blue)move fastest and elute first.IFractions are collected fromthe bottom of the column andanalyzed quantitatively.(b)(a)Figure 5—12 Ion-exchange chromatography. Anexample of a cation-exchange resin is presented.(a) Negatively charged sulfonate groups (—SO3 )on the resin surface attract and bind cations, suchas H~, Na", or cationic forms of amino acids.(b) An acidic solution (pH 3.0) of the amino acidmixture is poured on a column packed with resinand allowed to percolate through slowly. At pH 3.0the amino acids are largely cations with net positive charges, but they differ in the pKa values oftheir R groups, and hence in the extent to whichthey are ionized and in their tendency to bind tothe anionic resin.
As a result, they move throughthe column at different rates.jJJOLJUJLiJ10i_iI15I20I25I301Time (min)Figure 5-13 Automatically recorded high-performance liquid chromatographic analysis of aminoacids on a cation-exchange resin. The area undereach peak on the chromatogram is proportional tothe amount of each amino acid in the mixture.35Part II Structure and Catalysis124oIIOCOHxC+coo-H3N-C-HOH0NinhydrinH0^+ixII^HOONinhydrinAmino acidCO20oO/:=N-C.I3H2O + H +OO"Purple pigmentOne of the most important, technically and historically, is the ninhydrin reaction, which has been used for many years to detect andquantify microgram amounts of amino acids.
When amino acids areheated with excess ninhydrin, all those having a free a-amino groupyield a purple product. Proline, in which the a-amino group is substituted (forming an imino group), yields a yellow product. Under appropriate conditions the intensity of color produced (optical absorbance ofthe solution; see Box 5-1) is proportional to the amino acid concentration.
Comparing the absorbance to that of appropriate standard solutions is an accurate and technically simple method for measuringamino acid concentration.Several other convenient reagents are available that react with thea-amino group to form colored or fluorescent derivatives.
Unlike ninhydrin, these have the advantage that the intact R group of the aminoacid remains part of the product, so that derivatives of different aminoacids can be distinguished. Fluorescamine reacts rapidly with aminoacids and provides great sensitivity, yielding a highly fluorescent derivative that permits the detection of nanogram quantities of aminoacids (Fig. 5-14). Dabsyl chloride, dansyl chloride, and l-fluoro-2,4dinitrobenzene (Fig. 5-14) yield derivatives that are stable underharsh conditions such as those used in the hydrolysis of proteins.HH RU INO 2 + H - N - C —H HNO 2l-Fluoro-2,4-dinitrobeRzeneR H)NO 22,4-Dinitrophenylamino acida-Amino acidH R1+ I+ H—N—C—COO"HFluorescamineFigure 5—14 Reagents that react with the a-aminogroup of amino acids. The reactions producing 2,4dinitrophenyl and fluorescamine derivatives areillustrated. The reactions of dansyl chloride anddabsyl chloride are similar to that of l-fluoro-2,4dinitrobenzene (Sanger's reagent).
Because the derivatives of these reagents absorb light, theygreatly facilitate the detection and quantification ofthe amino acids.Ha-Amino acidFluorescentamine derivativeCH 3 CH 3NCH 3N=N-/NCH,SO2C1Dansyl chlorideV-SO2C1\ =Dabsyl chloridePeptidesWe now turn to polymers of amino acids, the peptides. Biologicallyoccurring peptides range in size from small molecules containing onlytwo or three amino acids to macromolecules containing thousands ofamino acids.
The focus here is on the structure and chemical propertiesof the smaller peptides, providing a prelude to the discussion of thelarge peptides called proteins in the next two chapters.Chapter 5 Amino Acids and PeptidesPeptides Are Chains of Amino Acids125H R2R1Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide.Such a linkage is formed by removal of the elements of water from thea-carboxyl group of one amino acid and the a-amino group of another(Fig.
5-15). Peptide-bond formation is an example of a condensationreaction, a common class of reaction in living cells. Note that as shownin Figure 5-15, this reaction has an equilibrium that favors reactantsrather than products. To make the reaction thermodynamically morefavorable, the carboxyl group must be chemically modified or activatedso that the hydroxyl group can be more readily eliminated. A chemicalapproach to this problem is outlined at the end of this chapter (see Box5-2). The biological approach to peptide bond formation is a majortopic of Chapter 26.Three amino acids can be joined by two peptide bonds to form atripeptide; similarly, amino acids can be linked to form tetrapeptidesand pentapeptides.
When a few amino acids are joined in this fashion,the structure is called an oligopeptide. When many amino acids arejoined, the product is called a polypeptide. Proteins may have thousands of amino acid units. Although the terms "protein" and "polypeptide" are sometimes used interchangeably, molecules referred to aspolypeptides generally have molecular weights below 10,000.Figure 5-16 shows the structure of a pentapeptide. The amino acidunits in a peptide are often called residues (each has lost a hydrogenatom from its amino group and a hydroxyl moiety from its carboxylgroup). The amino acid residue at that end of a peptide having a freea-amino group is the amino-terminal (or N-terminal) residue; theresidue at the other end, which has a free carboxyl group, is the carboxyl-terminal (C-terminal) residue.
By convention, short peptidesare named from the sequence of their constituent amino acids, beginning at the left with the amino-terminal residue and proceeding toward the carboxyl terminus at the right (Fig. 5-16).Although hydrolysis of peptide bonds is an exergonic reaction, itoccurs slowly because of its high activation energy. As a result, thepeptide bonds in proteins are quite stable under most intracellularconditions.The peptide bond is the single most important covalent bond linking amino acids in peptides and proteins. The only other type of covalent bond that occurs frequently enough to deserve special mentionhere is the disulfide bond sometimes formed between two cysteine residues. Disulfide bonds play a special role in the structure of many proteins, particularly those that function extracellularly, such as the hormone insulin and the immunoglobulins or antibodies.I IH.N-CH—C—OH + H—N-CH—COCTOH2O+R1H R2II IH 3 N-CH~C—I*—CH-COO"Figure 5—15 Formation of a peptide bond (shadedin gray) in a dipeptide.
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