Electrophoresis R. D.Strickland Research Service, Veterans Administration Hospital, Albuquerque, N. M .
This review extends the coverage of a previous one (227) by citing articles published between the latter half of 1969 and the end of 1973. The areas of discussion will be biased toward significant developments in methodology and theory. Applications are mentioned only for their illustrative value. Where possible, this deficiency is compensated for by references to books or reviews. METHODS AND APPARATUS Particle Electrophoresis. Continuous free curtain electrophoresis has been used to separate 16 classes of lymphoid cells (275), subcellular particles ( 1 9 3 , and microsomes (113). Both the methodology (98) and the limitations (266) of the method have been reviewed. Factors affecting the mobility of red cells have been discussed (226). There is a method and apparatus for preparing fractions of live lymphocytes (3). Microscopic Electrophoresis. Methods and apparatus for observing fractionations under a microscope have been developed for use with microorganisms (94) and bacterial or mammalian cells (146). Nucleic acid components were separated on a single cellulose fiber (241). The hemoglobin variants in a single erythrocyte have been separated (48). A micro disc method makes it possible to analyze the proteins from a single cell (79). Immunoelectrophoresis. A booklet describes the techniques of immunoelectrophoresis (6). The general methodology of immunoelectrophoresis in gels has been discussed extensively in a series of articles (164, 268, 269). An article in this series also describes immunoelectrophoresis on cellulose acetate membranes (138). Articles on methodology for special purposes include one describing the use of immunoelectrophoresis for detecting bacterial proteases (167) and one that describes the location and quantitation of antigens and antibodies ( 8 ) . Another article deals with the physical-chemical aspects of immunoelectrophoretic analysis in relation to antigens and antibodies (136). Two reviews discuss the methods used for diagnosing and detecting paraproteins (142, 205). Two-dimensional immunoelectrophoresis, also called Laurel1 cross-electrophoresis, can be used to obtain estimates of the amounts of proteins in a mixture such as serum (81). A sophisticated method for calculating quantities of precipitate using peak height as a parameter, gives the amounts of proteins with a mean error of &.5% (157). Including a reference standard, carbamoylated human transferrin, in the Laure11 system enhances the accuracy of quantitation (261). In a simplified method of two-dimensional electrophoresis, the antigens are caused to migrate into a pattern and are then made to travel a t right angles to the original path of migration into a bed containing antiserum. In this application, the precipitates form triangular peaks whose area can be measured by multiplying half the width of their base line times the height of the peak (247).The methodology for two-dimensional immunoelectrophoresis has been described in detail (9) Simultaneous electrophoresis of samples at two dilutions makes it possible to measure samples in which the components differ greatly in concentration (181). Cellulose acetate gels supported on sheets of mylar are easier to use and require less time for preparation than the gels conventionally used in crossed electrophoresis (162, 180). When antigens migrate in a gel containing specific antiserum, precipitates form that are shaped like rockets. The heights of the precipitates are proportional to the amount of antigen (262). All serum proteins with a mobility greater than y-globulins can be quantitated by this method. In order to quantitate y-globulins, it is necessary to alter their mobilities by carbamoylating them with cyanate (260). It is also possible to use carbamoylated antibodies; this has the advantage that
it does not require chemical modification of the antigens. This method can be used for measuring both polyclonal and monoclonal immune globulins (19). It is sometimes advantageous to separate proteins by conventional electrophoresis before submitting them to crossed immunoelectrophoresis. This is done by disc electrophoresis on polyacrylamide gel followed by electrophoretic transfer of the fractions into agarose for crossed immunoelectrophoresis (47, 85). In a simpler form of immunoelectrophoresis, electrophoresis is carried out in a polyacrylamide gel and the components are transferred to an agarose gel containing specific antiserum. In accomplishing this, it was necessary to reduce the electroendosmotic properties of the agarose gel by incorporating methyl cellulose (121). Evaporation from a gel surface causes a flow of water and solute through the gel. This has been called rheophoresis. This effect has been used to bring antigen into contact with antibody. The amount of reagents brought together is three times greater than is attained by the conventional method of diffusion through the gel. This greatly increases the sensitivity of detection (245). A novel method for performing immunoelectrophoresis, apparently developed independently by two groups (155, 276, 277), involves casting acrylamide gel into the form of a hollow cylinder by placing a plastic rod in the middle of a tube. Electrophoresis is carried out along the length of the cylinder with the rod still in place. When separation is complete, the rod is removed and antiserum is used to fill the cylinder. This causes the antigens to precipitate in clearly separated bands. The pattern obtained in this way is much easier to interpret than the overlapping arcs seen in conventional immunoelectrophoresis. Other detection reagents, such as stains, can be used instead of antiserum. Isoelectric Focusing. The most interesting development in electrofractionation during the past four years has been in the area of isoelectric focusing. The subject has been extensively reviewed (34, 95, 187, 192, 193, 250, 251, 264, 273). The method depends upon the commercial availability of a product called Ampholine (LKB-Produkter). This product is a mixture of aliphatic aminocarboxylic polymers ranging in molecular weight between 300 and 600 and having a wide range of isoelectric points. In a voltage gradient, the components of Ampholine arrange themselves in stationary bands in the order of their isoelectric points. Sample substances of migrating in this milieu cease to move when they arrive a t regions corresponding to their own isoelectric points. Since this is a new method, it is not surprising that apparatuses for isoelectric focusing have been developed in great profusion. These include microscale apparatuses consisting of capillary tubes filled with acrylamide gels made up in Ampholine (75, 80, 86, 92) and an apparatus making use of gel slabs (219, 249). In one application, serum proteins are first separated by electrofocusing in acrylamide-filled glass tubes and then submitted to electrophoresis in a vertical gel slab. The ability of these combined methods to resolve serum proteins is remarkable; approximately 90 separate spots were observed when the gel was stained with Coomassie Brilliant Blue (58). Sephadex C - 7 5 beds made by casting Ampholine suspensions of Sephadex on plastic plates have been used successfully to separate 20 isoenzymes of horseradish peroxidase (52) and more than 20 fractions of both metmyoglobin and oxymyoglobin in preparations from horses and sperm whales (185). Electrofocusing in free solution has the obvious advantage of allowing the operator to obtain fractions separate from a supporting medium, but this is difficult because of the disturbances arising from convection. A new apparatus is claimed to overcome these difficulties (242). One approach to stabilizing pH gradients has been to form them
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in sucrose gradients (231). Stable pH gradients have also been obtained in a small polyethylene tube coiled around a copper tube which serves to carry coolant. The small diameter of the tube permits ejecting the fractionated contents without mixing them. The apparatus can be used on a semi-preparative scale (152). Another apparatus in which a larger tube is bent in a serpentine curve can be used for preparative work; as much as one gram of protein can be fractionated in a single run (196). There is an arrangement whereby both the p H and UV-absorption of the effluent from a density gradient apparatus can be measured simultaneously. Both measured variables are recorded automatically (209). One worker has been occupied with applying instrumental methods to observing isoelectric focusing phenomena. The basis of his apparatus is a quartz tube in which isoelectric focusing is performed in free solution. The quartz tube permits automatic ultraviolet scanning of the developing pattern (29). A device for filling and emptying the column was also developed (30). Examples of the apparatus' use include fractionation of oligopeptides containing tyrosine and tryptophane (35), and of glycinine subunits in sucrose density gradients containing urea and dithiothreitol (37).The apparatus makes it possible to scan proteins a t various ultraviolet wavelengths while they are being focused (36). The scanning apparatus was attached to a digital on-line data acquisition system which was used to calculate the resolving power and zone resolution automatically (32, 33). The data that are automatically acquired and processed include changes in the position of peaks, peak areas, the segmental pH gradient, and the isoelectric points of proteins (31).It is claimed that the opportunity that this apparatus provides to observe isoelectric zones during the process of their formation, that is to say in the transient state, contributes greatly to the value of the data. Protein stains tend to react with the carrier Ampholine and so to interfere with the location of protein zones in polyacrylamide gel. This makes direct measurement of the proteins by ultraviolet scanning very useful (68). However, after isoelectric focusing, polyacrylamide gels containing protein zones can be successfully stained with Coomassie Brilliant Blue (248), or with Bromophenol Blue in 50% ethanol containing 5% acetic acid. The gel is destained with 30% ethanol, 5% acetic acid (7). The determination of isoelectric points by isoelectric focusing is facilitated by using amphoteric dyes as internal markers (44). The tendency of proteins to precipitate in Ampholine when they reach the zones of their isoelectric points can be overcome by the use of nonionic detergents ( 7 3 , or by the addition of ethylene glycol to the gel (123).Immunoelectrophoretic methods can be applied to identifying antigens after isoelectric focusing (27, 88). It is reported that the protein bands can be immobilized by treatment with gluteraldehyde after soaking the pattern in 18% sodium sulfate. This treatment does not affect the binding activity of antibodies (129). Gradients for isoelectric focusing in the pH range 2-5 can be made by adding acids to the commercially available carrier ampholyte (222). It is claimed that commercial bacto-peptone solutions yield a contiguous pH gradient over the range p H 3 to 9. If this is true, bactopeptone may be an inexpensive substitute for Ampholine in many applications (21). Another report states that isoelectric focusing of proteins can be accomplished without the use of special buffers. Unlike Ampholine which requires many hours to stabilize into an isoelectric gradient, a gradient is obtained within 10 seconds by using a temperature gradient to control pH. In the apparatus described, a continuous temperature gradient ranging between 0 and 50" was used. The pH gradient that results extends over one pH unit. The isoelectric points of the fractions focused in this apparatus are determined by means of thermistors inserted into the isoelectric zones. The apparatus has been extensively used to separate hemoglobins in 16 minutes (148). A pH gradient in Ampholine migrates during the process of electrofocusing. A polymerizing mixture containing methyl sulfoxide helps to prevent ampholyte shifting (15). Owing to the formation of local zones of high concentration, the conductivity of an ampholyte gradient in acrylamide gel is not uniform (74). Inorganic ions such as phosphate and carbonate in a gradient cause pH jumps in isoelectric fo96R
cusing gradients on polyacrylamide gel (20). It is important to place the protein in an isoelectric gradient somewhere near its isoelectric point. The recommended range is within one and one-half pH units. Failure to do this greatly reduces resolution (147). The presence of urea has a significant effect upon the isoelectric points of some proteins (124, 240). It is necessary to be aware of the possibility of interactions of Ampholine with proteins. Such interactions have been observed with liver alkaline phosphatase (144). bovine serum albumin (199, 258) and yeast isocitrate dehydrogenase (114). In addition to binding to the proteins, Ampholine can modify them by oxidizing cystine and methionine to cystic acid and methionine sulfoxide (116). Ampholine also interacts with the isozymes of yeast isocitrate dehydrogenate, causing a large apparent variation in the isoelectric point (114). Ferric myoglobin is reduced by Ampholine during acrylamide gel electrofocusing. The reduction is more rapid in gels polymerized by riboflavin than by persulfate. The difficulty can be obviated by adding potassium ferricyanide to the gel (183). Only one report of the use of pH gradient in a way comparable to preparative electrophoresis in a free buffer stream could be located (210). This lack may reflect the inherent difficulty of maintaining gradients in a moving liquid. Perhaps a pH gradient induced by maintaining a thermal gradient could be used. Most isoelectric focusing on the preparative scale has been done in large blocks of polyacrylamide gel (71, 230), large columns (224), or free solution with the liquid stabilized either by sucrose gradients (76),or mechanically (132). Once proteins are fractionated by isoelectric focusing, they can be freed of the ampholyte by precipitation with ammonium sulfate. The precipitates must be washed several times to free them from ampholyte with remains bound to the protein. The ampholyte components are not precipitated by ammonium sulfate (170).
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CONVENTIONAL ELECTROPHORESIS Apparatus. A method of electrofractionation that is only remotely related to electrophoresis has been described. It will be discussed here because of its potential importance. In this apparatus, a thin sheet of liquid is made to flow between two plates. Voltage is applied to the plates in order to produce a static electrical field. Charged molecules or ions in the flowing liquid move laterally toward the plate that bears an opposite charge; this retards their progress relative to the solvent flow. The tendency to diffuse away from a plate opposes the electrostatic attraction. The resultant of the two forces differs for each species of molecule, so different rates of movement in the direction of liquid flow occur. Proteins have already been separated by this method (26). Several improved apparatuses for use with starch gel have been described (12, 82, 109). There are several new techniques for performing electrophoresis in polyacrylamide gel cast on microscope slides (87, 161). One method of slide electrophoresis incorporates albumin into an agarose gel as an aid to separating lipoproteins. It is thought that the albumin reacts with the free fatty acids in the serum. Lipoproteins bind to agarose when free fatty acids are present (63).An apparatus for two-dimensional electrophoresis in polyacr lam ide permits separating the ribosomal proteins from Zsche: richia coli into 50 components (127). Continuous preparative electrophoresis can be accomplished in polyacrylamide gels (251, or in free flowing films of buffer (229). The zones in free films can be sharpened by controlling the zeta potential of the walls of the flow cell by electrical means (228). Continuous fractionation by combining electrophoresis with molecular sieving has been used to prepare human serum proteins and enzymes (53). Many new designs for batch preparative apparatus have been suggested; they make use of gels as supporting media and provide means for eluting the fractions sequentially (54, 105, 128, 190, 203, 214, 225, 234). New devices designed for use in electrophoresis include an optimum power supply ( Z O l ) , a system that cools thermoelectrically by using Peltier elements (137), a system for controlling and monitoring temperature (22), and an apparatus that permits rapid dialysis of microliter volumes of samples (117).
Richard D. Slrickland is a research biochemist at the Veterans Administration Hospital in Albuquerque, N.M.. He was originally employed by this institution, as clinical chemist, upon receiving his Ph.D. from the University of New Mexico in 1953. He has published extensively in the area of analytical biochemistry. A number of his papers have been concerned with electrophoresis as applied to the separation and measurement of proteins. At this time, he is investigating the modes of interaction with polyvalent cations of proteins, peptides, and amino acids.
Media. Polyacrylamide gel (28) is superior to both starch and agar for haptoglobin typing (104). The optimum concentration of acrylamide for fractionating RNA has been determined (176). Acrylamide gels can be modified to make them soluble by substituting N&"-diallytartramide for methylenebisacrylamide ( 4 ) ; they can be made to contain ion exchange groups by incorporating acrylic acid (122). Continuous concentration gradient rods (217) or slabs (5, 145) of polyacrylamide have been used to separate proteins on the basis of their molecular weights. Persulfate artifacts can be avoided by removing the persulfate electrophoretically (178) or by incorporating an antioxidant into the gel (130). Agarose has been modified by cross-linking it with epichlorohydrin, and by linking it to ion-exchange groups (202). Commercial agaroses from different sources differ greatly in their ability to separate mucoproteins (110). Charge-free agar can be prepared by desulfating the NaBH4 followed by reduction with LiAlH4 (141). Alginates make useful supporting gels (206, 207). A new cellulose gel (Cellogel RS) has been developed for hemoglobin electrophoresis (221). Cellulose gels can be used advantageously for separating lipoproteins (17, 23) and in characterizing glucose-6-phosphate dehydrogenase (72). Immunoelectrophoresis on cellulose acetate films instead of agar conserves samples and is much easier to perform (179). Gelatin has been neglected as a supporting gel because of its low melting point. This can be corrected by hardening the gelatin with formaldahyde. When this is done, the gelatin becomes an excellent support for immunoelectrophoresis (244). Owing to the enhanced effect on separation of molecular sieving, electrophoretic beds made of Sephedex gel are coming into widespread use (83, 118, 134, 236, 252, 267). The separation of seromucoid on starch-agar gel can be greatly improved by using starch that has been acetylated to remove its free hydroxyl groups. The technique for acetylation is easy (173). Pevicon is useful for preparative fractionation in columns (18, 263). The sensitivity of electroimmunodiffusion and of two-dimensional immunoelectrophoresis is increased by a factor of 5 by adding Dextran T-70 or polyethylene glycol to the gels containing the antibody (139). Buffers a n d Solvents. A number of discontinuous buffer systems have been proposed (171, 184). Several discontinuous systems have been developed for use with different kinds of cellulose aceeate (163). A computer program is available for generating the fnrmulas and properties of 4269 discontinuous buffer systems (125). The development of 3 new amine buffers together with those already available provide a series with pK's at 0.5-unit intervals in the pH range between 6.1 and 8.1 (42). Special buffer systems have been developed for separating plant gums and mucilages (238), wheat proteins (200), oligonucleotides (140), and myelin proteins (64).Twenty buffer systems for use in starch gel for the electrophoresis of enzymes have been tabulated (211). It is frequently necessary to solubilize cellular proteins prior to electrophoresis. This can be done with an acidified solution of phenol and urea or with 8M urea containing P-mercaptonethanol (100). Urea solutions completely solubilize the hormones of the pituitary gland (274). Mixtures of acetic acid, urea, phenol, and mercaptoethanol can be used to dissolve cell membranes (186). Neither urea nor sodium dodecyl sulfate produce subunits
when mucous glycoproteins are deaggregated (107j. The 11-S subunit of soybean globulin undergoes carbamoylation when it is incubated with urea in Tris buffer. This causes the appearance of artifact bands during gel electrophoresis (133). Sodium dodecyl sulfate has been very widely used to solubilize proteins. Examples include mycoplasma protein (49), wheat gliadins ( 6 5 ) , cell protein membranes (66), erythrocyte membranes (67), proteins of retina photoreceptors (213), cell membrane proteins (165), glomerular basement membranes substances ( 1 6 4 , polypeptides from zymogen granule membranes (151), ribonucleic acids (99), and envelope proteins from Escherchi coli (108). The nonionic detergent Triton X-100 is useful for solubilizing membrane proteins prior to their identification by immunoelectrophoresis (52). It should be noted that sodium dodecyl sulfate can form artifactual precipitin lines in immunoelectrophoresis (172). A nonionic detergent such as Tween 80 or Triton X-100 can be used to detect aggregated proteins by comparing the mobility of the proteins in their presence with the mobility of the same proteins in their absence. Both detergents cause a disaggregation that can be reversed by dialysis (215). Detection Methods. Techniques for detecting tritiumtagged RNA by autoradiography following polyacrylamide electrophoresis have been described (2, 39). A number of ways for preparing polyacrylamide gel for autoradiography are available (50, 89, 111, 243). An automated system for detecting radioactive zones in a polyacrylamide has been developed (10). Ferritin can be tagged with iodine-131 without altering its electrophoretic behavior in gel, but its mobility on paper is altered (120). The methods for liquid scintillation counting of macromolecules in acrylamide gels have been reviewed (93). Two new techniques for accomplishing this have been described (103, 175). Using visible dyes as markers for determining the length of migration during an electrophoretic run allows runs to be terminated a t a point determined by the distance of dye migration. Since the relative mobilities of the dye and the peptides are not altered by temperature, the loss of peptides by migration off the paper can be avoided (223). Polysaccharides can be stained with cyanuric chloride-Amido Black 10B prior to electrophoresis. This enables progress during electrophoresis to be observed visually (177). An electrophoretic protein pattern in a polyacrylamide gel can be observed as a series of dark bands if the gel is formed between a quartz plate and a fluorescent glass plate and is illuminated by an ultraviolet lamp (62). The patterns of serum-protein on cellulose acetate can be observed by infrared absorption a t 6 pm; proteins displa a strong absorption while cellulose acetate does not absorg appreciably a t this wavelength (265). Fast green stains proteins in urea-containing acrylamide gels without metachromasia (90). Commercial Amido Blacks (CI 20470) are impure to chromatographic analysis. This causes proteins in electrophoretic patterns to stain with different colors (270). Arginine rich histones can be differentiated from other histones on the basis of their staining with bromophenol blue (13). Buffalo Black is a highly sensitive stain for histones (272). Histones (45) and other proteins (212) can be stained by reaction with dansyl chloride. This permits their detection and measurement by fluorescence (45). Techniques for demonstrating tryptophane, tyrosine, and carbohydrate proteins isolated in gels have been described (70). Glycoproteins can be detected and measured by using the periodic acid-Schiff reagent (106, 159). Glycoproteins can also be detected by staining with basic fuchsin (24) or Alcian Blue (259). Glycosaminoglycans can be detected and measured by means of Alcian Blue 8 GX (101), or by staining with Toluidine Blue (102, 112). Serum lipoproteins can be prestained with Sudan Black B (78, 135). It is desirable to acetylate the Sudan Black B before using it in this way because the acetylated dye has a much greater affinity for lipoproteins (96). Lipoproteins do not take up dyes uniformly so it is necessary to apply correction factors if accurate quantitation is to be obtained (59-61). A superior method for staining lipoproteins is to oxidize the electrophorogram with ozone and then to apply the Schiff reaction (16, 153). Phosphoproteins can be stained by a method that involves hydrolyzing the phosphate ester linkages with a dilute base in the A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 5 , APRIL 1974
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presence of ionic calcium. This causes calcium phosphate to precipitate. Treating the precipitates with ammonium molybdate yields an insoluble nitrophosphomolybdate complex that can be stained with methyl green (46). Neurophysins can be stained with aldehyde-fuchsin (166). A systematic study of staining techniques for RNA showed the best results with Pyronine Y; Toluidine Blue 0 was also satisfactory (156). Electrophoresis of serum on starch gel sometimes yields nonstaining white zones when stained with Amido Black. The white zones are RNA (235). It is possible to separate materials from tissue by electrophoresis. Examples of this include the separation of thyroid hormones from thyroid glands (150) and the separation of @-lipoproteins from aortic intima by causing them to migrate from the tissue into an antibody containing gel (218). One application of this principle makes use of continuous particle electrophoresis. Treponemia pallid u m can only be cultured in living tissue. Continuous particle electrophoresis of homogenates of such tissue makes it ossible to obtain a pure suspension of Treponemia paltihum (204) There are new books describing electrophoresis on cellulose acetate (40), polyacrylamide (160), and electrophoresis for clinical purposes (38). Methods for separating nucleic acids (174, 182, 246, 271), plant proteins (198), and proteins and nucleic acids (154) have been reviewed. The applications of diagonal electrophoresis (97) and of electrophoresis (188) in detecting heterogeneity have been discussed. MOLECULAR CONSTANTS Polyacrylamide gels are unique among supporting media because their properties can be varied precisely and within wide limits by controlling gel concentration and the degree of cross-linkage (131). These properties, along with completion of excellent systematic studies of the variables that affect electrophoretic migration and resolution (149, 191, 208, 2 3 7 , have made possible a useful mathematical and theoretical approach to the problems of zone electrophoresis. Most of these developments have been summarized in review (41). As a result of these advances, zone electrophoresis in polyacrylamide gel is now a valuable tool for measuring molecular constants. Molecular Weight Determinations. Proteins and peptides interact with anionic detergents such as sodium dodecyl sulfate (SDS) in which one protein molecule binds many molecules of the detergent. The ratio of protein to bound SDS is nearly constant (on a gram/gram basis) for most proteins, which is to say that the size of the complex aggregate is typically in direct proportion to the molecular weight of the protein that forms the complex (189). The charge-mass ratio of such complexes is also nearly constant because the large number of negative charges accrued with the SDS makes the intrinsic charge of the protein a negligible factor. Given these circumstances, it is clear that the electrophoretic mobility in free solution (Mo) of protein complexes would be virtually the same for all species of protein-SDS complexes. In a gel, however, the movement of a particle is retarded to a degree that depends upon the size of the particle and the porosity of the gel (I, 143, 195). These considerations make it evident that the rate at which a protein-SDS complex migrates through a gel will be inversely related to the molecular weight of the protein. Under experimental conditions, in an SDS containing gel of suitable porosity, the relative LITERATURE CITED
(1) Ahmad-Zadeh, C., Piguet, J. D . , Pathoi. Microbiol. 1970, 36, 305. (2) Amaldi, P., Anal. Biochem. 1972, 50, 439. (3) Andersson. L. C., Nordling, S..Hayry, P . . Cell. immunol. 1973, 8, 235. (4) Anker. H . S., FEBS (Fed. Eur. Biochem. SOC.)Lett. 1970, 7, 293. (5) Arcus, A. C . , Anal. Biochem. 1970, 37, 53. (6) Arquembobrg, P. C., Salvaggio, J. E., Bickers, J. N.. "Primer of lmmunoelectrophoresis, with Interpretation of Pathologic Human Serum Patterns," Ann Arbor Humphrey: Ann Arbor, Mich., 1970, 83 pp. (7) Awdeh, Z. L . , Sci. Tools 1969, 16, 42. (8) Axelsen, N. H., Bock, E., J. immunoi.
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mobilities ( R f) are inversely proportional to the logarithm of the protein molecular weights. When known proteins are used for comparison, the unknown molecular weight of a protein can be estimated with considerable accuracy. A set of suitable protein standards in the molecular weight range 6,600 to 360,000 has been proposed; these proteins are marked by l-dimethylaminonaphthalene-5-sulfonylation so that they can be observed under W-light (115).Polyacrylamide gel is usually used for measuring molecular weights because of the exactness with which pore size can be controlled by varying the degree of cross-linking and the acrylamide concentration. The properties of this gel can be varied in many ways to suit it to a specific application (41, 131). The picture presented above is idealized; some proteins behave anomalously because of cystine cross-linking which diminishes SDS binding (14, 91), because the samples, e.g., histones, oligopeptides (233) and the al- and cuz-chains of collagen (232) have high intrinsic charges or for steric reasons. The difficulties encountered have usually been overcome by using homologous proteins of known molecular weight as standards. Cystine cross-linking can be eliminated by reducing the protein (119) with dithioerythritol (253). Intrinsic charges can be changed by maleylation (239). The basic method for measuring molecular weight has been greatly refined: Relative mobilities ( R f ) are measured in a series of gels with a range of porosities caused by varyin the acrylamide concentration (7'). Plotting Log Rf us. #gives a linear plot whose slope has been named the retardation coefficient ( K r ) .Kr is directly proportional to molecular weight. The use of multiple gels permits detection of anomalous mobility behavior that would not be recognized in a single gel (169, 216). The use of several gel concentrations enables the measurement of molecular characteristics other than molecular weight. SDS is not needed for these measurements. Other Constants. Free solution mobility (Mo) is measured by extrapolating the Rf us. T lot to zero gel con(e.g., Pyronin-Y) centration. A substance with known is used to relate R f to Mo (11, 84, 194). Einstein-Stokes radii (rEs) are determined by finding the gel concentration that excludes a protein (exclusion limit). This is done by extrapolating an R f us. T plot to R, = 0. Plotting known rEs against experimentally determined exclusion limits gives a straight line that can be used to find rEs for an unknown (69, 194, 256). One variation in the method of measuring molecular radii makes use of molecular sieving in Sephadex gel beds. The proteins are separated by electrophoresis in thin layers of Sephadex and then caused to migrate by rheophoresis (evaporative flow) which is equivalent to chromatography. The retention coefficient for a molecule varies as a linear function of its hydrodynamic radius (254, 255, 257). The isoelectric point of a protein can be measured by finding its mobility in several gels made up in buffers that differ in pH. Plotting mobility against pH and extrapolating to zero mobility gives an accurate estimate of PI (43, 126). Estimating PI by isoelectric focusing is probably easier. The techniques described here have been used to measure the molecular weights and other properties of nucleic acid (220), polynucleotides (55, 56, 73) and low molecular weight DNA fragments (57). The nucleic acid (220) was measured in a nonaqueous solvent (formamide) to eliminate conformational effects. Within each set of homologous pol mers, hondroitin sulfates A and B give a linear log Rf us. $plot (158).
Methods 1972, 1, 109. (9) Baessler, K. H., G i T (Glas-instrum.Tech.) Fachz. Lab. 1971,15,1261. (10) Bakay. B., Anal. Biochem. 1971, 40, 429. (11) Banker, G. A., Cotman, C. W., J. Biol. Chem. 1972, 247,5856. (12) Baron, J . C., Cah. ORSTOM (Off. Rech. Sci. Tech. Outre-Mer), Ser. Oceanogr. 1972, 10, 251. (13) Barrett, I . D . . Johns, E. W., J. Chromatogr. 1973, 75, 161. (14) Barton, R. J., Biochem. J. 1972, 129, 983. (15) , , Bates. L. S.. Devoe. C. W.. J. Chromatour. 1972, 73, 296. (16) Belanger, M . , Lapointe, J. R., Sobolewski. G , Lavai. Med. 1971, 42, 249. (17) Berends, G. T.. De Jong. J . . Zondag, H. A,, Clin. Chim. Acta 1972, 41, 187.
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Berti, G., Ventrelli, I., Boll. SOC. ltai. Biol. Sper. 1972, 48,499. Bjerrum, 0.J.. Ingild, A,, Lowenstein, H.. Weeke, B., Ciin. Chim. Acta 1973, 46, 337. Blaich, R., Naturwissenschatten 1971, 58, 55. Blanicky. P., Pihar, 0..Collect. Czech. Chem. Comrnun. 1972, 37,319. Biattler, D. P., Bradley, A. R . , Anal. Biochem. 1972, 47,296. Bonzel, J . , Bonzel, H., Feuiii. Bioi. 1969, 10,57. Borzynski, L. J., McDougall, W. J., Norton, D. A., Stain Techno/. 1972. 47, 317. Boyde, T. R. C., Remtulla, M . A , , Anal. Biochem. 1973, 55, 492. Caldwell. K. D . , Kesner. L. F., Myers, M .
(27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38)
(39) (40)
(41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53) (54)
N., Giddings, J . C., Science 1972, 176, 296. Carrel. S., Theilkaes, L., Skvaril, S., Barandun. s.,J. Chromatogr. 7969, 45, 483. Castilla, J., Villanueva. E., Gisbert-Calabuig, J. A,. Med. Leg. Domm. Corpor. 7972, 5, 52. Catsimpoolas. N., Anal. Biochem. 1971, 44,411. lbid., 7977, 44, 427. /bid., 1973, 54, 66. /bid., 1973, 54, 79. lbid., 1973, 54, 88. Catsimpoolas, N.. Separ. Sci. 1973, 8, 71. Catsimpoolas. N., Campbell, B. E., Anal. Biochem. 1972, 46,674. Catsimpoolas, N., Wang, J.. Anal. Biochem. 1971, 39, 141. lbid., 44, 436. Cawley, L. P.. Chin, H. P., Epstein, E., Kelsey, R . L., "Clinical Electrophoresis: A Manual," Gelman Inst. Co., Ann Arbor, Mich., 1969, 160 pp. Chia, Li-Li S. Y., Randerath. K., Randerath, E., Anal. Biochem. 1973, 55, 102. Chin, H. P., "Cellulose Acetate Electrophoresis. Techniques and Applications," Ann Arbor-Humphrey, Ann Arbor, Mich., 1970, 139 pp. Chrambach, A., Rodbard, D.. Science, 1971, 440. Clayton, J. W., Tretiak, D. N., J. fish. Res. Boardcan. 1972, 29, 1169. Cocola, F., Neri, P., Boll. SOC. /tal. Biol. Sper. 1971, 47, 818. Conway-Jacobs, A., Lewin, L. M . , Anal. Biochem. 1971, 43,394. Creighton, M. O., Trevithick, J. R., Anal. Biochem. 1972, 50,255. Cutting, J. A,, Roth, T. F , Anal. Biochem. 1973, 54, 386. Dames, W., Maurer, H. R., Neuhoff, V., Hoppe-Seyler's Z. Physiol. Chem. 1972, 353, 354. Dan. M., Exp. CellRes. 1970, 63, 436. Daniels, M . J . , Meddins, B. M., J Gen. Microbiol. 1973, 76, 239. Daniels, M . J., Wild, D.G., Anal. Biochem. 7970, 35, 544. Delincee, H., Radola. B. J., Blochim. Biophys. Acta 1970, 200, 404. Demus, H., Mehl. E., Biochim. Biophys. Acta 1970, 211, 148. Dietrich, C. P., Anal. Biochem. 1973, 51, 345. Diezel, W.. Kopperschlaeger, G., Hofmann, E., Acta Biol. Med. Ger. 1970, 24, 771
(55) Dingman, C. W., Eur. Biophys. Congr., PrOC., 1st. 1971. 1, 251 (56) Dingman. C. W.. Fisher, M . P., Kakefuda, T.. Biochemistry 1972, 11, 1242. (57) Dingman, C. W., Kakefuda, Fisher, M. P.. Anal. Biochem. 1972, 50, 519. (58) Domschke, W., Seyde, W., Domagk. G. F.. Z. Klin. Chem. Klin. Biochem. 1970, 8, 319. (59) Dyerberg, J . , Hjoerne. N.. Clin. Chim. Acta 7970, 28, 203. (60) lbid., 30, 407. (61) lbid., 1971, 33, 458' (62) Eisinger, J . , Biochem. Biophys. Res. Commun. 1971, 44, 1135. (63) Elphick, M. C., J. Clin. Pafhol. 1971, 24, 83. (64) Eng. L . F., Bond. P.. Gerstl, B., Neurobiology 1971, 1, 58. (65) Ewart. J A D . , J . Sci. Food Agr. 1973, 24, 685. (66) Fairbanks, G . , Avruch. J., J. Supramolecular Struct. 1972, 1, 66. (67) Fairbanks, G., Steck, T. L.. Wallach. D. F. H . , Biochemistry 1971, 10, 2606. (68) Fawcett, J . S.,Protides Biol. Fluids, Proc. Colloq. 1969, 17, 409. (69) Felgenhauer. K.. Clin. Chim. Acta 1971, 32, 53. (70) Felgenhauer. K . , Weis, A., Glenner, G. G., J . Chromatogr. 1970, 46, 116. (71) Finlayson, G. R . , Chrambach. A,, Anal. Biochem. 1971, 40,292. (72) Fiorelli. G., Quad. Sciavo Diagn. Clin. Lab. 1970. 6. 100. (73) Fisher, M P , Dinqman, C W , Biochemfstry 1971, 10, 1895(74) Frater, R , A n a l Biochem 1970, 38, 536 (75) Fredriksson, S , Anal B!ochem 1972, 50,
575. (76) Freedman, M . H.. J. Immunol. Methods 1972, 1, 177. (77) Friesen, A. D . Jamieson, J. C., Ashton, F. E.. Anal. Biochem. 1971, 41 149. (78) Frjngs, C. S..Foster, L. B., Cohen, P. S.. clm. Chem. 1971, 17, 111. 179) Gainer, H., Anal. Biochem. 7971, 44, 589. (80) lbid., 1973, 51, 646. (81) Gay, St.. Rosenkranz, M., Geiler, G., Deut. Gesundheitsw. 7971, 26. 1697. (82) Geldermann, H., Anim. Blood Groups Biochem. Gen. 1970, I,229. (83) Ghidalia, W., Vendrely, R., Bull. SOC. Chim. Biol. 1970, 52, 110. (84) Ghosh, S., Basu, M. K., Schweppe, J. S.. Anal. Biochem. 1972, 50,592. (65) Giebel, W., Saechtling, H., Hoppe-Seyler's Z. Physiol. Chem. 1973, 354, 673. (86) Godson, G. N., Anal. Biochem. 1970, 35, 66. (87) Goetz, H.. Scheiffarth, F., Eberl, M . . Z. Klin. Chem. Klin. Biochem. 1970, 8, 306. (88) Good, A. H.. Ceverha. B. B., J. lmmunol. 1971, 106, 1677. (89) Goodman, D., Matzura, H., Anal. Biochem. 1971, 42,481, (90) Gorovsky, M . A., Carlson, K.. Rosenbaum, J. L., Anal. Biochem. 1970, 35, 359. (91) Griffith, I. P., Biochem. J. 1972, 126, 553. (92) Grossbach, U., Blochem. Biophys. Res. Comm. 1972, 49,667. (93) Grower, M. F., Bransome, E. D., Jr., Curr. Status Liquid Scinfill. Counting 1970, 263. (94) Gusev. V. S., Zvyagintsev, D. G., Vestn. Mosk. Univ., Biol., Pochvoved. 1971, 26, 90. (95) Haglund, H., Methods Biochem. Anal. 1971, 19, 1. (96) Hall, F. F., Ratliff, C. R., Westfall, C. L., Culp, T. W., Biochem. Med. 1972, 6, 464. (97) Han, K., Dautrevaux, M., Biserte, G., Ann. Pharm. f r . 1972, 30, 379. (98) Hannig, K., Heidrich, H. G., Klofat, W., Pascher, G., Schweiger, A,, Stahn, R.. Zeiller, K., Tech. Biochem. Biophys. Morpho/. 1972, 1, 191. (99) Harewood, K.. Wolff. J. S.. Anal. Biochem. 1973, 55, 573. (100) Hastiz, M., Szeienyi. J. G., Hoilan, S. R., Baumann, M., Haematologia 1972, 6, 249. (101) Hata. R . , Nagai, Y., Anal. Biochem. 7973, 52, 652. (102) Havez, R., Degand, P., Boersma, A,. Richef, C., Clin. Chem. Acta 1971, 33, 443. (103) Hemminki, K., Acta Chem. Scand. 1971, 25,3887, (104) Hilgermann, R.. Z. Rechtsmed. 1972, 71, 222. (105) Hodson, A. W., Latner, A. L., Anal. Biochem. 1971, 41, 522. (106) Holden, K. G.. Yim, N. C. F., Griggs, L. J., Weisbach, J. A., Biochemistry 1971, 10, 3105. 107) lbid., 3110. 108) Holland, I . B., Tuckett, S., J. Supramolecular Struct. 1972, 1, 77. 109) Hori. S. H., Kamada. T., Yonezawa, S., Acta Histochem. Cytochem. 1971, 4, 107. 110) Houtsmuller, A. J.. Protides Biol. fluids, Proc. Colloq. 1969, 17, 523. 111) Howard, G. A,. Traut. R. R., f E B S ( f e d . Eur. Biochem. SOC.)Lett. 1973, 29, 177. 112) Hsu, D.. Hoffman, P.. Mashburn, T. A,, Anal. Biochem. 1972, 46, 156. (113) Ichishita, H., Nakayama. H., Ishida. A,, Tanaka, S., Kumamoto Med. J. 1972, 25, 83. (114) Illingworth, J. A , , Biochem. J. 1972, 129, 1125. (115) Inouye, M . , J. Biol. Chem. 1971, 246, 4834. (116) Jacobs, S.,Analyst (London), 1973, 98, 25. (117) Jacobson, K. 8.. Anal. Biochem. 1970, 38, 555. (118) Jaworek, D.. lnt. Symp. Chromafogr. Electrophor., Lect. Pap., 6th. 1970, 118. (119) Jeffrey, P. D . , Aust. J. Biol. Sci. 1970. 23, 809. (120) Johannsen, E.. Ges, J . , Knoll, P., Wiss. Z. Karl-Marx Univ. Leipzig, Math.-Naturwiss. Reihe 1969, 18, 593. (121) Johansson. B. W., Stenflo. J . , Anal. Biochem. 1971, 40,232. (122) Joice. J. R . , Klemperer, H. G.. Anal. Biochem. 1971, 41, 581
(123) Jones, R. E., Hemmings. W. A,, Faulk. W. P., lmmunochemistry 1971, 8, 299. (124) Josephson, R. V., Maheswaran, S. K., Morr. C. V., Jenness, R., Lindorfer, R. K.. Anal. Biochem. 1971, 40,476. (125) Jovin, T. K., Dante, M. L.. Chrambach. A,, "Multiphasic Buffer Systems Output," Federal Scientific and Technical Information. U.S. Department of Commerce, Springfield, Va., 1971. (126) Kaltschmidt, E., Anal. Biochem. 1971, 43, 25. 127) Kaltschmidt, E., Wittmann, H . G., Anal. Biochem. 1970. 36, 401, 126) Kawata, H., Chase, M. W., Elyjiw, R . , Machek, E., Anal. Biochem. 1971, 39, 93. 129) Keck, K., Grossberg, A. L., Pressman, D., Eur. J. lmmunol. 1973, 3, 99. 130) King, E. E., J. Chromatogr, 1970, 53, 559. 131) Kinqsbury, N., Masters, C. J., Anal. Bioc h e h 1970, 36.144. 132) Kiryukhin. I. F., Byull. Eksp. Biol. Med. 1972. 120. ~. 74. -~ 133) Kitamura, K., Okubo. K.. Shibasaki, K.. Agr. Biol. Chem. 1973, 37, 1983. (134) Klein, A., Chudzik, J., Sarnecka-Keller. M . . J. Chromatogr. 1970, 53, 329. (135) Klein, G. C., Cooper, G. R . , Stand. Mefhods Clin. Chem. 1970, 6, 127. (136) Kieist, H., Wiss. Z. Univ. Rostock, Math.Naturwiss. Reihe 1969, 18, 625. (137) Kleist, H., Friemel. H.. Brock, J., Muecke, D.. Nelius, H., Z. Med. Labortech. 1970, 11, 351. (138) Kohn, J., Methods lmmunol. lmmunochem. 1971, 3, 273. (139) Kostner, G., Holasek. A,, Anal. Biochem. 1972, 46, 680. (140) Krawczyk, A., Dziembor, E., Bull. Acad. Pol. SCi., Ser. Sci. Biol. 1971, 19, 81. (141) Laas, T., J. Chromatogr. 1972, 66,347. (142) Lamerz, R., Fateh-Moghadam, A,, fortschr. Med. 1971, 89, 332. (143) Lamy. J., Compin, S., Weill, J.. Ann. Biol. Clin. 1971, 29, 125. (144) Latner. A. L., Parsons, M. E., Skillen, A. W., Biochem. J. 1970, 118,299. (145) Laulhere. J. P., Lambert, J., Berducou. J. Analysis 1972, 1, 234. (146) Lemp. J . F., Jr., Asbury, E. D., Ridenour, E. O., Biotechnol. Bioeng. 1971, 13, 17. (147) Lewin, S., Blochem. J. 1970, 117,41. (148) Luner. S. J., Kolin, A,, Proc. Nat. Acad. Sci. U.S. 1970, 66, 898. (149) Lunney. J., Chrambach, A,. Rodbard, D., Anal. Biochem. 1971, 40, 158. (150) Lybeck. H., Leppaluoto, J.. Aito, H., Ann. Med. Exp. Biol. fenn. 1969, 47, 161. (151) MacDonald, R . J . , Ronzio. R. A,. Biochem. Biophys. Res. Commun. 1972, 49, 377. (152) Macko, V., Stegemann, H.. Anal. Biochem. 1970, 37, 186. (153) Magnani, H. N.. Howard, A. N., J. Clin. Pathol. 1971, 24, 837. (154) Maizel, J. V., Jr., fundam. Tech. Virol. 1969, 334. (155) Makonkawkeyoon, S., Hague, R . , Anal. Biochem. 1970, 36,422. (156) Marcinka. K.. Anal. Biochem. 1972, 50, 304. (157) Markowski, B., Clin. Chim. Acta 1973, 44, 319. (158) Mathews, M. B., Decker, L., Biochim. Biophys. Acta 1971, 244, 30. (159) Matthieu. J. M.. Ouarles. R . H., Anal. Biochem. 1973, 55,313. (160) Maurer, H. R., "Disc Electrophoresis and Related Techniques of Polyacrilamide Gel Electrophoresis" 2nd ed., de Gruyter. New York, N.Y., 1971, 222 pp, (161) Maurer, H. R . . Dati, F. A,, Anal. Biochem. 1972, 46, 19. (162) Miller, J. N., Mutzelberg, I . D.. J. Chromatogr. 1973, 75, 165. 1163) Miller. J. N.. Newton, A. H., Burns, D . T.. Clin. Chim. Acta 1971, 31, 427. (164) Minchin. C. H. G., Methods lmmunol. lmmunochem. 1971, 3,287. (165) Mira y Lopez, R., Siekevitz, P , Anal. Biochem. 7973, 53,594. (166) Moens, L., Burford, G. D , Anal. Biochem. 1973, 51, 466. (167) Mueller. H. E.. Zentralbl. Bakteriol. Parasitenk., lntektionskr. Hyg., Abt. 7: Orig., Reihe A 1972, 220, 466. (168) Myers, C.. Bartlett, P., Biochim. Biophys.
-.
~I
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 5, A P R I L 1974
99R
Acta 1972,290, 150. (169) Neville, D. M . , Jr.. J . Biol. Chem. 1971, 246,6328. (170) Nilsson. P., Wadstrom, T., Vesterberg. O., Blochim. Biophys. Acta 1970,1, 146. (171) Qrr, M . D., Blakley. R. L., Panagou, D., Anal. Blochem. 1971,45,68. (172) Palmer, E. L., Martin, M . L., Hierholzer, J. C., Ziegler, D. W.. Appl. Microbiol. 1971, 21,903. (173) Pankina, V. Kh.. Lab. Delo 1970 (3),183. (174) Parish, J. H., "Subcell. Components: Prep.
c. E., Schammel,
P., Thayer. J. D., Appl. Microbiol. 1970,19,287. (205) Schneider, W., Blut 1970,20,4. (206) Schvma. D.. Taubert. M.. Aerztl. Forsch ~I
1976,24,326. (207) /bid., 1971,25,68. (208) Scurzi, W., Fishbein, W. N . , Trans. N.Y. Acad. Sci. 1973,35,396. (209) Secchi, C., Anal. Biochem. 1973,51,448. (210) Seiler, N., Thobe, J., Werner, G., HoppeSeyler's Z. Physiol. Chem. 351,865. (211) Shaw. C. R . , Prasad, R., Biochem. Genet. 1970,4,297. Fractionation," 2nd ed., G. E. Birnie, Ed., Butterworths, London, 1972,p 251. (212) Shelton, K. R., Biochem. Biophys. Res, Commun. 1971,43,367. (175) Paus, P. N., Anal. Biochem. 1971, 42, 372. 1213) Shikolvukov. S. A,. Ukr. Biokhim. Zh 1973,45,55. (176) Paus, P. N., Alfheim, I., Anal. Biochem. 1972,50,430. (214)Shuster, L.. Methods Enzymol. 1971, 22, 434. (177) Pavlenko, A. F., Ovodov, Yu, S.,J. Chromatogr. 1970,52, 165. (215)Singh, J., Wasserman, A. R . , Biochim. Biophys. Acta 1970,221,379. (178) Petropakis, H. J., Anglemier, A. F., Montgomery, M . W., Anal. Biochem. 1972, 46, (216)Skakoun, A., Daussant. J.. Mayer, C., Bios. (Paris). 1972,3, 503. 594. (217) Smeds, S I Bjorkman, U , J Chromatogr (179) Pfaefflin, W., Med. Lab. 1970,23,85. 1972,71,499 (180) Pizzolato, M. A., Clin. Chim. Acta 1973, 45,207. (218)Smith E B , Slater, R S , Btochem J 1971 123.39 (181) Platt. H. S.,Sewell, B. M., Feldman, T., Souhami. R. L.. Clin. Chim. Acta 1973.46. (219)Soderholm, J., Allestam. P , Wadstrom, T.. FEBS (Fed. Eur. Bfochem. Soc.) Lett. 419. 1972,24,89. (182) Popescu, M.. Stud. Cercet. lnframicrobiob lnframicrobiol. 1971,22,475. (220)Stainov, D Z.,Pinder. J C Gratzer. W (183) Quinn. J. R.. J. Chromatogr. 1973, 76, B , Nature (London), New Biol 1972,235, 520. .-. 108. (184) Racusen, D., Foote, M., Anal. Biochem. (221)Stangalini. A,, Pedrinazzi. R . C., Del 1968,25, 164. Campo, G. B., Minerva Pediat. 1972, 24, 1460. (185) Radola, B. J., Biochim. Biophys. Acta 1969,194,335. (222) Stenman, U. H., Grasbeck, R . , Biochim. Biophys. Acta 1972,286,243. (186) Ray, T. K., Marinetti, G. V., Biochim. Biophys. Acta 1971,233,787. (223) Stevenson, K. J., Anal. Biochem. 1971, 40,29. (187) Reis, H. E., Wetter, O., Klin. Wochenschr. 1970,48,643. (224) Stewart-Tull, D. E. S., Arbuthnott. J. P., Sci. Tools 1971,18,17. (188) Ressler, N., Anal. Biochem. 1973, 51, 589. (225) Strauch, L., Ann. Biol. Clin. (Paris) 1971, (189) Reynolds, J. A,, Tanford, C., Proc. Nat. 29,229. Acad. Sci. U S . 1970,66,1002. (226) Streiff, F., Stoltz, J. F., Genetet, B.. Nouv. Rev. Fr. Hematoi. 1971,11, 913. (190) Reznick, A. Z.,Allen, H . J., Winzler. R. J., Anal. Biochem. 1973,52,395. (227) . , Strickland. R. D., Anal. Chem. 1970, 42, 32. (191) . . Richards, E. G., Lecanidou, R . , Anal. Biochem. 1971,40,43. (228) Strickler, A,, Sacks, T., Ann. N.Y. Acad. Sci. 1973,209,497. (192) Rilbe, H., Ann. N.Y. Acad. Sci. 1973, 209, 11. (229) Strickler, A,, Sacks, T., Prep. Biochem. 1973. (193) Rilbe, H., Protides Biol. Fluids, Proc. . 3. 769. Coloo. 1969.17.369. (230) Suzuki, T., Benesch. R . E., Yung, S., Benesch, R., Anal. Biochem. 1973,55,249. (194) Rodbard, D., Chrambach, A,, Anal. Bio(231) Svendsen, P. J., Protides Bioi. Fluids, chem. 1971.,40,95. Proc. Colloq. 1969,17,413. (195) Rodbard, D., Kapadia, G., Chrambach, A., Anal. Biochem. 1971,40,135. (232)Svojtkova, E., Deyl. Z.,Adam, M.. J. Chromatogr. 1973,84,147. (196) Rose, C., Harboe, N. M. G., Portides Biol. Fluids, Proc. Colloq. 1969,17,395. (233) Swank, R. T.. Munkres. K. D . , Anal. Biochem. 1971,39,462. (197) Ryan, K. J., Kalant, H., Thomas, E. L., J. CellBiol. 1971,49,235. (234) Szvlit, M.. lnt. Symp. Chromatogr. Electrophoresis, 5th 1968,121 (198)Safonov, V. I., Safonova, M. P., Biokhim. Metody Fiziol. Rast. 1971, 113. (235)Tata, S J , Edwin, E E , J. Rubber Res lnst Malava 1969 21.477 (199) Salaman, M. R . , Williamson, A. R . , Biochem. J. 1971,122,93. (236)Tedesco. ?. A,, Eonow, R . , Mellman, W. J.. Anal. Biochem. 1972,46,173. (200) Sasek, A,, Rostl. Vyroba 1970,16,1303. (201) . . Schatter, H . E., Johnson, F. M., Anal. Bio- (237)Thorun, W.. 2. Klin. Chem. Kiin. Biochem. chem. 1973,51,577. 1971,9,3. (202) Schell, H . D., Ghetie, V., Rom. Biochem. (238)Tomoda, M., Akiyama, T . , Shoyakugaku Zasshi 1969,23,64. Lett. 1970, 103. (203) Schilling, E., Horn, A,, Boernig, H.,Acta (239)Tung, J.. Knight, C. A,. Biochem. Biophys. Res. Commun. 1971,42,1117. Biol. Med. Ger. 1972,29,341. (204) Schmale, J. D . , Kellogg, D. S..Jr.. Miller, (240) Ui, N.,Biochim. Biophys. Acta 1971, 229, ~i
- I
l00R
ANAL.YTlCAL CHEMISTRY, VOL. 46, N O . 5, APRIL 1974
567. (241) Uzorin, E. K., Shungskaya, V. E.. Tsitologiya 1969,11, 1064. (242) Vglmet, E., Protides Bioi. Fluids, Proc. Colloq. 1969,17,401 (243)Van Dongen, C. A. M.. Peil. P. S. M., Anal. Biochem. 1973,53,654. (244) Van Orden, D. E., lrnmunochemistry 1971, 8,869. (245) Van Oss, C. J., Bronson, P. M.. lmmunochemistry 1969,6,775. (246) Vasu, S., Stud. Cercet. lnframicrobiol. 1971,22,459. (247) Versey. J. M. B., Slater, L., Ann. Clin. Biochem. 1973,10,1 , (248) Vesterberg. O., Biochim. Biophys. Acta 1971,243,345. (249) lbid., 1972,257, 1 1 , (250) Vesterberg, O.,Methods Enzymoi. 1971, 22,389. (251)Vesterberg, O.,Protides Biol. Fluids, Proc. Coiloq. 1969,17,383. (252)Virella, G.. Experienfia 1973,29,502. (253)Virella, G., Parkhbuse, R. M . E.. lmmunology 1972,23,857. (254)Waldmann-Meyer, H., Biochim. Biophys. Acta 1972.261,148. (255) Waldmann-Meyer H., Eur. Biophys. Congr., Proc., 1st 1971,1, 159. (256) Waidmann-Meyer, H . , Protides Bioi. Fluids, Proc. Colloq. 1968,16,715. (257) lbid., 1969,17,527. (258) Wallevik, K., Biochim. Biophys. Acta 1973,322,75. (259) Wardi, A. H., Michos, G. A,, Anal. Biochem. 1972,49,607. (260)Weeke. B., Aerztl. Lab. 1972,18,12. (261)Weeke, B., Scand. J. Clin. Lab. Invest. 1970,25, 161. (262)Weeke B., Ugeskr. Laeger 1969, 131, 1419. (263) Weiss, J. B., Brenchiey, P., Biochem. SOC.Trans. 1973,1, 571. (264)Wellner, D., Anal. Chem. 1971,43,59. (265) Wenzel, M., Hoffmann. K..2. Kiin. Chem. Ciin. Biochem. 1973,1 1 , 270. (266) West, C. R., Augustin, R., Cytol. Automat., Proc. Tenovus Symp. 2nd. 1968, 121. (267) Whitehead, J. S.,Kay, E., Lew, J. Y . , Shannon, L. M., Anal. Biochem. 1971,40, 287. (268) Williams, C. A,, Methods immunol. lmmunochem. 1971,3,234. (269) lbid., 237. (270)Wilson, C. M., Anal. Biochem. 1973, 53, 538. (271) Wojcicki, J., Postepy Biochem. 1970. 16, 551. (272) Wray. W., Stubblefield, E . , Anal. Biochem. 1970,38,454. (273)Wrigley, C. W., Methods Enzymol. 1971, 22,559. (274) Zanini, A., Giannattasio, G.. J. Endocrinoi, 1972.53,177. (275) Zeiller. K., Pascher, G., Hannig, K.. Prep. Biochem. 1972,2,21 (276)Zeinih, R . A,, Mbawa, E . , Pillay, V. K. G.. Fiorella. B. J.. Dunea. G.. Biochim. Biophys Acta 1973,317,1 (277) Zeinih, R A , Mbawa, E Pillay V K G , Fiorella, B J , J Lab Cifn Med 1973,82,
326
