2 Структура и функция белка (1160071), страница 9
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Because a molecule of water (Mr 18) isremoved to create each peptide bond, the average molecular weight ofan amino acid residue in a protein is about 128 — 18 = 110. Table 6-1shows the number of amino acid residues in several proteins.Proteins Have Characteristic Amino Acid CompositionsAs is true for simple peptides, hydrolysis of proteins with acid or baseyields a mixture of free a-amino acids.
When completely hydrolyzed,each type of protein yields a characteristic proportion or mixture of thedifferent amino acids. Table 6-2 shows the composition of the aminoacid mixtures obtained on complete hydrolysis of human cytochrome cand of bovine chymotrypsinogen, the inactive precursor of the digestiveenzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind ofamino acid they contain.
The 20 amino acids almost never occur inequal amounts in proteins. Some amino acids may occur only once permolecule or not at all in a given type of protein; others may occur inlarge numbers.Some Proteins Contain Chemical GroupsOther Than Amino AcidsMany proteins, such as the enzymes ribonuclease and chymotrypsinogen, contain only amino acids and no other chemical groups; these areconsidered simple proteins.
However, some proteins contain chemicalcomponents in addition to amino acids; these are called conjugatedproteins. The non-amino acid part of a conjugated protein is usuallycalled its prosthetic group. Conjugated proteins are classified on thebasis of the chemical nature of their prosthetic groups (Table 6-3); forexample, lipoproteins contain lipids, glycoproteins contain sugargroups, and metalloproteins contain a specific metal. A numberof proteins contain more than one prosthetic group. Usually theprosthetic group plays an important role in the protein's biologicalfunction.Table 6-3 Conjugated proteinsClassProsthetic groupExampleLipoproteinsGlycoproteinsPhosphoproteinsHemoproteinsFlavoproteinsMetalloproteinsLipidsCarbohydratesPhosphate groupsHeme (iron porphyrin)Flavin nucleotidesIronZincCalciumMolybdenumCopperft-Lipoprotein of bloodImmunoglobulin GCasein of milkHemoglobinSuccinate dehydrogenaseFerritinAlcohol dehydrogenaseCalmodulinDinitrogenasePlastocyaninTable 6-2 Amino acid compositionof two proteinsNumber of residuesper molecule of proteinAminoacidAlaArgAsnAspCysGinGluGlyHisHeLeuLysMetPheProSerThrTrpIVrValTotalHumancytochrome cBovinechymotrypsinogen62532281338618334271532241581010523210191426928238423104245138Part II Structure and CatalysisWorking with ProteinsThe aggregate biochemical picture of protein structure and function isderived from the study of many individual proteins.
To study a proteinin any detail it must be separated from all other proteins in a cell, andtechniques must be available to determine its properties. The necessary methods come from protein chemistry, a discipline as old as biochemistry itself and one that retains a central position in biochemicalresearch. Modern techniques are providing ever newer experimentalinsights into the critical relationship between the structure of a protein and its function.Proteins Can Be Separated and PurifiedCells contain thousands of different kinds of proteins. A pure preparation of a given protein is essential before its properties, amino acidcomposition, and sequence can be determined.
How, then, can one protein be purified?Methods for separating proteins take advantage of properties suchas charge, size, and solubility, which vary from one protein to the next.Because many proteins bind to other biomolecules, proteins can also beseparated on the basis of their binding properties. The source of a protein is generally tissue or microbial cells. The cells must be brokenopen and the protein released into a solution called a crude extract.
Ifnecessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles (see Fig. 2-24). Once the extract ororganelle preparation is ready, a variety of methods are available forseparation of proteins. Ion-exchange chromatography (see Fig. 5-12)can be used to separate proteins with different charges in much thesame way that it separates amino acids. Other chromatographic methods take advantage of differences in size, binding affinity, and solubility (Fig.
6-2). Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures.The approach to the purification of a "new" protein, one not previously isolated, is guided both by established precedents and commonsense.
In most cases, several different methods must be used sequentially to completely purify a protein. The choice of method is somewhatempirical, and many protocols may be tried before the most effective isdetermined. Trial and error can often be minimized by using purification procedures developed for similar proteins as a guide. Publishedpurification protocols are available for many thousands of proteins.Common sense dictates that inexpensive procedures be used first,when the total volume and number of contaminants is greatest.
Chromatographic methods are often impractical at early stages because theamount of chromatographic medium needed increases with samplesize. As each purification step is completed, the sample size generallybecomes smaller (Table 6-4) and more sophisticated (and expensive)chromatographic procedures can be applied.Individual Proteins Can Be QuantifiedIn order to purify a protein, it is essential to have an assay to detectand quantify that protein in the presence of many other proteins.Often, purification must proceed in the absence of any informationabout the size and physical properties of the protein, or the fraction ofthe total protein mass it represents in the extract.rvey.Protein ofinterestLigandAMixtureof proteinsLigand coupledto polymerbead• VSolutionof ligandPorouspolymer beadsProtein mixture is addedto column containingcross-linked polymer.Protein mixtureis added tocolumn containinga polymer-boundligand specificfor protein of1 2 3 4 5interest.Protein molecules separateby size; larger moleculespass more freely, appearingin the earlier fractions.I123 45 6Unwantedproteinsare washedthroughcolumn.(a)(b)Table 6-4 A purification table for a hypothetical enzyme*Procedureor step1.
Crude cellularextract2. Precipitation3. Ion-exchangechromatography4. Size-exclusionchromatography5. Affinity chromatographyFractionvolume(ml)Totalprotein(mg)Activity(units)1,40028010,0003,000100,00096,000329040080,0002008010060,0006006345,00015,000Specific activity(units/mg)10* All data represent the status of the sample after the procedure indicated in the first columnhas been carried out.OD3 4 5 6Proteinof interestis eluted byligand solution.Figure 6-2 Two types of chromatographic methodsused in protein purification, (a) Size-exclusion chromatography; also called gel filtration.
This methodseparates proteins according to size. The columncontains a cross-linked polymer with pores of selected size. Larger proteins migrate faster thansmaller ones, because they are too large to enterthe pores in the beads and hence take a more direct route through the column. The smaller proteins enter the pores and are slowed by the morelabyrinthian path they take through the column.(b) Affinity chromatography separates proteins bytheir binding specificities. The proteins retained onthe column are those that bind specifically to a ligand cross-linked to the beads. (In biochemistry,the term "ligand" is used to refer to a group or molecule that is bound.) After nonspecific proteins arewashed through the column, the bound protein ofparticular interest is eluted by a solution containing free ligand.140Part II Structure and CatalysisThe amount of an enzyme in a given solution or tissue extract canbe assayed in terms of the catalytic effect it 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 the reaction products, (3) whether the enzyme requirescofactors such as metal ions or coenzymes, (4) the dependence of theenzyme activity on substrate concentration, (5) the optimum pH, and(6) a temperature zone in which the enzyme is stable and has highactivity.
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