Table 111.
Determination of Purity
of Alpha, Beta-Unsaturated Compounds b y Bromination
Average Purity, Wt. Bromination Acrylic acid 98.4 f 0 . 2 (3) Butyl acrylate 98.0 i 0 . 1 (4) Cellosolve acrylate 94.5 i 0 . 1 (2) Crotonic acid 99.7 f 0 . 1 (4) Dibutyl fumarate 99.8 f 0 . 0 (2) Dibutyl maleate 96.5 i 0 . 1 ( 2 ) Diethyl fumarate 99.4 i 0 . 1 (2) Diethyl maleate 98.3 i 0 . 1 (2) Ethyl acrylate 99. o e Ethyl crotonate 97.6 f 0 . 1 (2) Fumaric acid 99.2 i 0 . 1 (3) Maleic acid 98.6 It 0 . 1 (3) Methyl methacrylate 9 9 . 5 i 0 . 1 (3) a Figures in parentheses represent number of detns. Morpholine method (3). Sodium sulfite method (2). d Saponification. Std. dev. for 15 degrees of freedom is 0.11. f Total acidity.
in Table I11 show that for most of the compounds studied, a precision to *O.l% was obtained. I n the case of ethyl acrylate the standard deviation for 15 degrees of freedom was 0.11. The method has been used for the determination of the purity of maleic and fumaric esters. The unsaturation
Other 98. 5b 98.4b 95.46 99.9c 99.v 97. 2d 98. 7 c 98. 7c 99. O b 98. Ob 99.9f 99. Of 99.5b
of these compounds is difficult to obtain by other methods. The morpholine method (4) can be used if a conductometric titration is employed. This requires a special instrument and is timeconsuming. The sodium sulfite method (3) is applicable to only the lower esters because of solubility difficulties.
I n general, compounds that interfere in the Kaufmann bromine-bromide method will interfere in this procedure. Large quantities of alcohols may interfere. Primary alcohols will interfere in the determination of esters but not in that of acids. Secondary alcohols and aldehydes are oxidized by bromine. Many of the interferences due to oxidation or substitution can be inhibited by conducting the bromination a t 0' C. LITERATURE CITED
(1) Beesing, D TV., Tyler, W. P., Kurta,
D. M . , Harrison, S. A., ANAL. CHEM. 21, 1073 (1949). (2) Byrne, R. E., Johnson, J. B., Zbid., 28, 126-9 (1956). (3) Critchfield, F. E., Johnson, J. B., Zbzd., 28,73 (1956). (4) Zbid., 28, 76 (1956). (5) Kaufmann, H. P., Z. Untersuch. Lebensna. 51, 3 (1926). (6) Lucas, H. J., Pressman, David, I N D . E N G . CHEhf., ANAL. ED. 10, 140 (1938). (7) Rosenmund, K. TV., Kuhnheim, W., Rosenherg-Gruszynski, D., Rosetti, H., Z. Unterszich. Nahr. u. Genussm. 46, I54 119231. (8) Williams, G., Trans. Faraday SOC.37, 749 (1941). RECEIVED for review November 13, 1958. Accepted April 24, 1959.
Dye-B ind ing Ca pacities of EIeven Electrophoretically Separated Serum Proteins R. D. STRICKLAND, T. R. PODLESKI, F. T. GURULE, M. L. FREEMAN, and W. A. CHILDSI Research Division, Veterans Administration Hospifal, Albuquerque,
b The use of dye-binding capacities for estimating serum proteins following their separation by electrophoresis in agar has been made possible by the availability of a gravimetric method suitable for measuring dye uptake of micro amounts of proteins. When 1 1 electrophoretically separated serum proteins were tested as to their abilities to bind Amido Black 1 OB, bromophenol blue, and Ponceau 2R, not only did the various fractions behave differently toward the different dyes, but corresponding protein fractions from different sera varied widely in their abilities to bind the same dyes. Large errors can result from dependence on this method for estimating electrophoretically separated serum proteins.
pipcr
rlectrophoresis, proteins of serum usually estiiimtcd by staining electrop1iorogr:Im and measuring &Y:mpmwiit,
1408
0
ANALYTICAL CHEMISTRY
the are the the
N. M.
amount of dye that associates with each fraction (2, 3, 6). This method would be satisfactory if all serum proteins had equal dye-binding capacities: actually, they differ in their affinities for dye, so that some workers (1, 3, 4) have determined proportionality factors to bring amounts of protein estimated by dyeing into agreement with those estimated by the classical methods. Staining has not been evaluated as a means for measuring the amounts of proteins separated by electrophoresis in agar. The availability of a gravimetric method (6) makes possible the direct measurement of the dye-binding capacities of serum protein fractions. This paper reports a n investigation of the staining properties of 11 serum proteins with respect to three commonly used dyes.
Caucasian males whose ages ranged between 24 and 45 years. The serum proteins were separated by electrophoresis in agar using the apparatus and techniques as described (5). The protein fractions were located and prepared as described (5) and before staining, each protein precipitate was washed once with 5 ml. of 10% trichloroacetic acid. The proteins were stained by suspending the precipitates in 2 ml. of a dye solution. Three dyes were tested: Ponceau 2R (National Aniline Division, Allied Chemical Corp.), Amido Black 10R (Hartman-Leddon Co., Inc.), and bromophenol blue (Hartman-Leddon Co., Inc.). The dye solutions were prepared by dissolving I00 mg. of a dye in 100 ml. of a snlvent consisting of 10% acetic acid, 45% methanol, and 45y0 distilled water (v./v.). The proteins remained in contact with the dye solu-
METHODS
1 Present address, Department of Surgwy, University of Colorado School of Medicine, Denver, Colo.
Sera Fere contributed by six healthy
tion for 48 hours or more. During the staining period the tubes were capped with Parafilm (Marathon Corp.) and kept in a refrigerator a t 4 " C. The dyed protein precipitates were separated by centrifugation. Excess dye was removed by two washings with 2-ml. portions of a solution containing lOyo acetic acid, 45% methanol, and 45y0 water (v./v.), then the precipitates were resuspended in wash solution, and collected by filtration through microcrucibles (6). The dyed protein precipitates were brought to constant weight a t 100" C. and weighed on a microbalance. The amount of dye bound to each fraction was determined spectrophotometrically after redissolving the protein-dye complex in 0.1N sodium hydroxide. A Beckman Model B spectrophotometer equipped with Corex cuvettes was used. Amido Black 10B and bromophenol blue were measured a t 590 mp; Ponceau 2R a t 460 mp. I n this alkaline solution the absorbance maxima of these dyes were unaffected by the associated protein. The distribution of bound dye among the various fractions was calculated by dividing the amount of dye associated with a given fraction by the sum total of dye bound to all fractions from an electrophorogram. The m i g h t of protein in each precipitate was calculated by subtracting the spectrophotometrically estimated weight of bound dye from the weight of the protein-dye complex. From these weights, the distribution of the fractions v a s calculated by dividing the weight of protein in each fraction by the total w i g h t of protein precipitated from an electrophorogram. For comparison. separate electrophorograms of each serum aere prepared and the distribution of the undyed precipitates was determined by the direct gravimetric method (6). Factors for estimating the m i g h t of protein from the amount of bound dye were carculated for each fraction by dividing the weight of protein by the weight of associated dye.
>
e 31
RESULTS AND DISCUSSION
The percentage distributions of the three dyes among the various fractions and the distributions of the gravimetrically determined associated proteins are shown in Table I. The distribution, as calculated from direct gravimetric determinations of fractions from undyed electrophorograms, is also given for comparison. When appropriate fractions are pooled on the basis of mobilities, the dye distribution percentages are in good agreement with those reported for the five protein fractions separated by paper electrophoresis (1, 3, 4 ) . Of the three dyes, the results from the use of bromophenol blue agree best with the gravimetric protein percentages. This apVOL. 31, NO. 8, AUGUST 1959
1409
Table II.
Fractions Rho Albumin-1 Albumin-2 Alpha1 Alpha*-1 Alphar2 Beta Gamma-1 Gamma-2 Gamma-3 Gamma-4
Dye-Binding Factors of Serum Proteins
(Six normal sera) Mg. Protein/Mg. Dye Std. Dev. Amido Black Bromophenol Ponceau 10B blue 2R 2.57 f 0.29 3.90 f 0.03 3.54 f 0.63 2.69 f 0.53 4.83 f 0.19 2.54 i= 0.48 3.24 f 0.63 4 . 1 5 f 0.18 4.27 f 1.29 3.47 f 0.82 5.26 f 0.69 4.81 f 1.54 3.82 i 0.88 4.96 f 0.55 5.31 f 1 . 5 4 3.78 f 1.04 4.65 f 0 77 5.11 i 1.20 3.66 f 1.24 4.78 f 1.62 4.15 f 0.75 3.84 f 0 73 3.84 f 1.62 5.84 f 1.57 3.90 ==! 1.10 4.60 Z!Z 1.12 5.30 + 1 . 2 3 3.79 f 0.90 5.42 f 1.01 5.10 i 1.22 4.81 f 1.31 2.30 f 3.60 7 . 7 6 i 3.82
parent advantage of bromophenol blue is probably due to its nonspecific staining properties. This dye has a n unfortunate tendency to fade in alkaline solution, so that large errors result unless the dye is determined very quickly after the precipitate has been dissolved. The distributions of Ponceau 2R and Amido Black 10B roughly approximate the distributions of the basic amino acids among the fractions. The apparent agreement between percentage of protein as determined gravimetrically and by staining is to a great
extent due to the tendency for individual differences to disappear when a n average is taken. The wide range of variation between the amount of protein in a fraction and the amount of associated dye is shown in Table I1 \There the protein-dye ratios (dyebinding factors) for the various fractions are given. The large standard deviations of the factors show that disagreements can be expected betiyeen determinations of protein by gravimetry and by a dyeing method. The wide variations in dye-binding capacity that
occur, not only among the different fractions from the same serum, but among corresponding fractions from different sera, make it evident that the method of dyeing can be expected to detect only gross abnormalities in serum protein distribution. LITERATURE CITED
(1) Fuchs, W., Flach, *4,, Hila. Wochschr. 33,903-6 (1955). (2) Grassmann, W., Hannig, K., 2. physiol. Chem. Hoppe-Seyler’s 290, 1-27 (1952). (3) Jenks, W. P., Jetton, M. R., Durrum, E. L., Biochern. J . 60,205-15 (1955). (4) Kusunoki, T., J . Biochem. ( T o k y o ) 40, 277-85 (1953). (5) Strickland, R. D., Mack, P. A., Gurule, F. T., Podleski, T. R., Salome, O., Childs, W. A , , ANAL.CHEW31. 1410 (1959). (6) Strickland, R . D., Podleski, T. R., Am. J . Clin. Pathol. 28, No. 4 (1957). RECEIVEDfor review January 26, 1959. Accepted April 15, 1959. Investigation supported in part by research grant H2100 from the National Heart Institute of National Institutes of Health, Public Health Service. From the Department of Surgery, University of Colorado School of Medicine, Denver, Colo., and the Surgical and Biochemical sections of the Veterans Administration Hospital, Albuquerque,
x. AI.
Determining Serum Proteins Gravimetrically after Agar Electrophoresis R. D. STRICKLAND, P. A. MACK, F. T. GURULE, T. R. PODLESKI, 0. SALOME, and W. A. CHILDS Research Division, Veterans Administration Hozpital, Albuquerque,
b An apparatus for electrophoresis in agar and a novel micromethod for estimating proteins gravimetrically have been developed. Eleven protein components in normal human serum are determined and this information is used to establish normal concentration ranges. Kjeldahl nitrogen factors are determined for each of the fractions and shown to vary significantly both among the different fractions and between the same fractions from different individuals. This makes possible the direct determination of complex serum proteins which contain prosthetic groups thai cannot b e estimated by the Kjeldahl method. This work underscores the fact that the division of serum proteins into five fractions is purely arbitrary and essentially meaningless.
T
use of agar gel to suppress convection currents which interfere with electrophoretic separations is gainHE
14 10
ANALYTICAL CHEMISTRY
N. M.
ing favor ( 1 , 5 , 6 ) . Agar gel gives better fractionation and permits larger sample volumes than does filter paper; furthermore, the separated fractions are easier to locate than when opaque materials such as powdered glass ( 2 ) or starch ( 3 ) are used as supporting media. The use of agar for electrophoresis has been limited by operational difficulties and by the lack of an accurate method for measuring the fractions. This paper describes the construction of an easy-to-use electrophoresis apparatus and gives the details of a method for determining protein fractions gravimetrically. This method has been used to investigate normal variations in the concentrations of 11 protein fractions from human serum and the results have been collated with concomitant microdeterminations of Kjeldah1 nitrogen. CONSTRUCTION A N D USE OF APPARATUS
The electrophoresis apparatus (Figure l ) , constructed entirely of acrylic plastic,
is a box divided bj- a vertical n d l into two equal, electrically isolated compartments. Each compartment is subdivided into interconnecting cells by partitions as high as the central w-all but which have 0.5-cm. gaps between their lower edges and the floor of the apparatus. Adjacent to the gaps, toward the center of the apparatus, are low nalls that reach slightly higher than the gaps. When the apparatus is to be used, the gaps between the cells are closed by pouring a 2% solution of agar in buffer into the end cells until the level of the agar stands a t the tops of the low walls. Once this agar has set, the end cells are filled complctely with the agar solution and the inside cells are filled with buffer. The agar barriers thus formed provide electrical bridges between the electrodes through the electrophoresis bed while serving as mechanical obstructions to the diffusion of electrode products. These barriers may be used repeatedly until they became contaminated with mold. The agar supplied by Baltimore Riological Laboratory, Inc. (Catalog S o . 02-106) can be used without purifica-
