Owen A. Moe, Jr. and Gary Smith Lebanon Valley College Annville, Pennsylvania 17003
Determination of the Molecular Weight and Subunit Stoichiometry of a Protein A biochemistry laboratory experiment
Many proteins exist in their native state a s tightly associated complexes of multiple, independently folded plypeptide chains. T h e name given t o this type of protein is oligomer, while the individual component polypeptide chains are referred t o a s protomers or subunits. This associated subunit structure, which usually does not involve any interchain covalent bonds, is defined a s t h e quaternary structure of the protein. Experimental methods t o measure the molecular weight of an oligomeric protein and t o determine the number and size of its comoonent subunits are essential to the biochemist. Such measurements are most accurately accomnlished thmueh the use of an analvtical ultracentrifuee. For " most undergraduate laboratories, however, this type of instrumentation is too exoensive and so~histicatedt o he of practical value. A simole but reasonablv accurate method to measure the molecular weight and subunit composition of a n oligomeric protein is described in the experiment preaented here. This experiment, which involves a n~mbinationof sodium dndecyl sulfate gel t:lectrophoresis and chemical cross-linking techniques was adapted for use in an undergraduate biochemistry course from a literature report by ihvies and Stark ( I ). Polysrrylnmide gel rlcctrophareri,, carrwd uut in the presence of sod~umdodreyl xdfate, can he used to determine the autwnit molecdar weights uf pnmins (21. Sodium dodecyl sultbte (SDS) is a detercent wh~rhhmlv hydn~phol~irslly to proteins in a large and relatively
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constant amount of -1.4 grams per gram of protein (3). The detergent-protein interaction has the effect of denaturing the noncovalent quaternary structure of the protein, causing a release of the individual polypeptide chains in the form of SDS-polypeptide particles which are rod-like in shaoe. The leneth and size of these narticles are nronortinnal to the mdecular weyeht of the .oolvnentide chains (3;. . . In n ,~ . addirion, the WS-pulgpep~idrpnrrwles passers a large, uniform negative charge which is due tv ihr SDS sultlmir acid r r ~ i d u and ~s which effectively overcomes the charge contribution of the polypeptide side chains. Polyacrylamidegel electrophoresis can effect a separation of proteins on the basisof charge tomass ratia as wellas size ( 4 ) ,the latter effect beine due to a sievine action arisinefrom the cross-linked eel matrix. ln'the presence O ~ S D Sfor , reasons stated above, all paTypeptides have nearly the same charge to mass ratio., Electrophoretic separation is accomplished, therefore, predominantly on the basis of polypeptide size (molecular weight). Empirical plots of log molecular weight versus electrophoretic mobility yield linear relationships for most proteins (2). hi molecular weight range of effective separation is determined by the extent of cross-linking of the gel (3). Plots of mobilitv versus loe molecular weieht for moteins of known molecular weights can serve as standard curves for unknown proteins. This
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method yields subunit molecular weights with an accuracyof 5-lW (3). The molecular weight of an oligomer, as well as its subunit stoichiometry, may be determined if chemical cross-linkingof the protein molecule is used in conjunction with SDS polyacrylamide gel electrophoresis (I). Protein cross-linkingis accomplished through the use of bifunctionalreagents which contain two reactive groups separated by an inert chain or ring structure. Thereagent employed in this experiment was dimethylsuberimidatewhich reacts with protein amino groups in an amidation reaction as outlined below (5).
II I
C-(CHJ6--C OCH,
II
+
bCH,
Protein
\
NHs
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392 1 Journal of Chemical Education
h this figurethe logarithm of the molecular weight of known protein standards (hexagons)is plotted versus electrophoretic mobility, me protein Standards are described in the text. The mobilities of the bands resuitino - from the eiectmphoretic analysis of crosslinked hemoglobin are indicated by the arrows.
A Summary of Student Data for the Determination of Hemoglobin
Molecular Weight and Subunit Stoichiametrv M~leculsr
Protein Mobility
Weigh@ Exoecfed
Band
average
average
1
0.90 0.66 0.48 0.36
15.000 t 1,600 29.000 f 2.600 47.000 f 2.500 63.000 f 4.000
2 3 4
aExorerred ar an deviation.
average of four
15.50031.00046,50062.000-
MW
erimidate and 1 mg of hemoglobin are added to 1.0 ml of 0.20 M triethanolamine (or triethylamine) and the pH is immediately adiusted to the ranae of a 9 with sodium hvdroxide and DH indicator baoer. The reaction is carried out for 90 min a t room temoerature and ihen auenehed bv the addition of 1.0 ml of a denaturntion huffer if! ..--. -1 M sodium phosphate, 1% sodium dodecyl sulfate, 1%2-mercapto ethanol, pH 7.0). After incubation in the denaturation buffer for 2 hr a t 37-C, the cross-linked hemoglobin is stored frozen a t -20'C. The lolhwing protein, are suggested as moleculnr wight standardc: lywzyme ( I l.INl,trypsin ~?:I,:ll~t),,lartatedehgdrogrnase ~ . l t i , ~ u ~ l . ovnlburnin (4:r.Nb). catahre !R2.0001. and bovine wrum nlhumm (68,000). The proteins are incubated in denaturation buffer for 2 hr a t 37% a t a concentration of 0.5 mglml and then stored a t -20°C prior to electrophoresis. Standard techniques (2,3)may be used in the preparation of 7.5% ~
monomer dimer trlmer tetramer
sets of students' data, f standard
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This reaction mav involve nratein amino erouns which reside an .. the Pame or on diNcrent protomrr rhaim of a giwn oligomrric prbcein molecule. Thus, for example, ifa native tetramer is allow~dto rewc uith dimethvl~ubertmidate,thendenatured wrthS1)Sand analgmd by polyacrylamide gel electrophoresis, protein bands corresponding to the monomer as well as the covalently cross-linked dimer, trimer, and tetramer species would be found. Intermolecular reactions hetween amino "erouns molecules. which would . from different olieomer " produce crow-linked species ul molazular weight greater than the termrnrr,mn br avoid4 by proper expcrimenul runtrol ultht. prowin concentration in the reaction mixture (1). Determination of the molecular weights of the four cross-linked species which are separated eleetrophoretieally may then be determined through the use of the molecular weight standard curve described above. This wocedure allows verification of the Dmwr subunit stoichiometty and measurement of the molecular weight of the native oligomer. The protein used for the experiment presented here is hemoglobin, a tetramer of molecular weight 62,000 (6). Hemoglobin iscomposed of two types of polypeptide chains, a and 8, such that the native hemoglobin molecule has the structure ad&. Although the a and @chains differ in both amino acid sequence and composition (6), their molecular weights are very nearly identical (15,500 glmole) (2). Hemoglobin was chosen for this experiment because it is commercially available in a pure form a t a low price. Experimental Materiab Hemoglobin, Comassie Brilliant Blue R, 2-mereaptaethanol, and the proteins used as molecular weight standards were purchased from Sigma Chemical Corporation. Acrylamide, methylenebisaerylamide, ammonium persulfate, and N,N,N',N'-tetrarnethylenediamine, used in the preparation of gels, were obtained from E-C Apparatus. Suberonitrile was purchased from Aldrich Chemical Company. Eleetrophoresis grade sodium dodecyl sulfate was purchased from Bio-Rad Laboratories. All other chemicals are commonly available. A Model 150A electrophoresis cell from Bio-Rad laboratories was used with a Gelman power supply. Procedures Dimethylsuherimidate may be synthesized from suberonitrile according toDavies and Stark (I). Anhydrous HC1 is bubbled through a solution of 0.5 g of suberonitrile in 2 ml of dried methanol (over Linde molecular sieve) and 15 ml of anhydrous ether for 30 mi". The reaction mixture is stored a t 4-C for 24 hr and the product is then precipitated hy the addition of 10 ml of anhydrous ether. After filtration, the product is washed well with a 1:3 ("1") mixture of dry methanol and ether. The dimethvlsuberimidate mav.. he stored a t 2.5%: over desirrnnt and is stnhle over a year's dwatiun. Fur the hemuclohm crops.linking reaction. 2 mg of dimethylsub~
hemoglobin or protein standard solutions. Fifty-microliter samples of each protein are layered on the gels and electrophoresis is carried out for 3-5 hr a t 8 mA per gel. The reservoir buffer is 2.8 X 10-2 M sodium dihydrogen phosphate, 7.2 X M disodium hydrogen phosphate, and 0.1% sodium dodecyl sulfate. The migration of the bromophenol blue tracking dye is marked by cutting a notch or placinga wire in the gel and the protein bands arestained overnight in a solution of 1.25gComassie Brilliant Blue R i n 227 mlmethanol, 227 ml water, and 46 ml of elacial acetic acid. Destainine is aecomplished by diffusion using a solution of 50 ml methanol, 75 ml glacial acetic acid, and 875 ml of water. Results a n d Discussion The electrophoretic migration of each protein hand is measured from its advancingedge and its mobility expressed as a fraction, relative to the migration of the traekingdye. Triplicate mobility determinations for the cross-linked hemoglobin and each protein standard may be accomplished during a single electrophoresis, using a cell with a capacity of 12 gel positions. This requires application of several different protein standards per gel. Student data, used to construct a plot af log molecular weight versus mohility, are shown in the figure. The corresponding mobilities of each of the four hemoglobin bands are indicated by the arrows in the figure. A summary of four different sets of data from a semester laboratory course is oresented in the table.
not occur under the conditions used in the crosr-linking reaction. Even when the hemoglobin concentration was increased 2-3 fold, only a very small amount of higher molecular weight material was found on thegels. Themolecular weights for the hemoglobin hands in the table were well within the 5-10% accuracy limits for the predicted values for monomer. dimer. trimer. and tetramer.
useful techniques of polyacrylamide gel electrophoresis and chemical modification reactions of proteins. Literature Cited
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Volume 54, Number 6.June 1977 1 393
