Table II.
Fractions Rho Albumin-1 Albumin-2 Alpha1 Alpha*-1 Alp har2 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 4.15 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 1.54 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!= 1.12 5.30 + 1.23 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 AND 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-
5101 t c a l l o w electrical contocl with agar b a r r i e r
Platinum
electrodes
LOCKING
RING
-
TOP V I E W
FUNNEL T O P
I 2 C T
i 1 ~
~3 0 :m
7 r J+
/ / E n d of c h a m b e r r e m o v e d t c show ccnsrruclion
Figure 1 .
Apparatus for electrophoresis in agar
A S S E MB L E D A P P A R A T U S SUPPORTING
BASE
Figure 2.
tion fur any of the applications described. The support for the electrophoresis strips is made by milling channels to cwnnect two parallel slots a t opposite ends of a rectangle of inch thick pldstic. The slots are cut entirely through the plastic; when the support is in place, they rest upon the agar 1)ariiera a t the end$ of the apparatus, nlloming electrical continuity between the inner cells of the apparatus when the channels are filled with agar. Samples are prepared by soaking blocks of 1% agar measuring 6 X 10 X 2 mm. in serum a t 4" C. for 24 hours, which is enough time to equilibrate the protein concentration of the blocks with that of the serum. For electrophoresis, the sample blocks are blotted free of adherent liquid and placed centrally in separate channels which then are filled ITith a lyOsolution of agar in buffer. This agar solution should be cooled to 46" C. before pouring to avoid protein denaturation. The agar should he poured a t once after the samples have been placed; delay results in the formation of semipermeable membranes around the blocks due to superficial drying. When the agar sets, the entire apparatus is covered with Saran Wrap (a thin plastic film), transferred to a 4" C. refrigerator, and connected through platinum electrodes in the inner cells to a direct current power supply. The separations were obtained in diethyl barbiturate buffer of 0.1 ionic strength a t p H 8.6 by applying a 2.5volt per cm. potential gradient between the channel ends for 24 hours. After electrophoresis, the agar strips are lifted from their channels by a long, narrow, stainless steel spatula and transferred to a bath of 10% trichloroacetic acid. I n half an hour the protein fractions become clearly visible as turbid
Holder for microcrucibles
Dimensions, locking ring, 6.5 X 2 cm.; funnel top, 4.5 supporting base, 5.5 X 3.5 cm.
areas in the agar, Each fraction is cut from the strip, placed in a 15-nil. centrifuge tube, covered with 2 ml. of water, and heated in a boiling nater bath until the agar has melted. Two milliliters of 2OY0 trichloroacetic acid are added, and the contents of the tube are mixed by lateral shaking, and then allowed to cool. The precipitate is packed firmly by centrifugation, the supernatant liquid discarded, and the tube allowed to drain into absorbent paper. The protein precipitate is washed twice with 10% trichloroacetic acid, then transferred by filtration to microcrucibles. The filtration apparatus (Figure 2), constructed from acrylic plastic, consists of a funnel top, a supporting base for the crucibles, and a locking ring to hold the funnel firmly against the supporting base. A thin rubber gasket prevents leakage when crucibles are clamped between the funnel and the base. During use the apparatus rests on a rubber ring fastened to the top of a suction flask. The microcrucibles are made by cutti?g f/& disks from No. 12 gold foil with a cork borer. The crucibles are shaped by placing the disks on the supporting base of the filtration apparatus and applying gentle suction. A steel needle is used to make a few tiny perforations near the center of the crucibles. Filter mats of acid-washed asbestos are built up in the crucibles in the usual \Yay. Crucibles so prepared weigh no more than 10 mg. A Cahn electrobalance (Cahn Instrument Co.) used for weighing is capable of weighing microgram amounts with good accuracy in less than a minute. The
X 2.5 cm.;
small load limit of this balance is effectively offset by using the lightweight crucibles. Protein precipitates are collected by resuspending them in 10% trichloroacetic acid and filtering the suspension through crucibles which have been brought to constant weight by heating to 100" C. After filtration the precipitates are brought to constant LTeight by drying a t this temperature. During pilot experiments, drying temperatures were varied between 60" and 125" C. without measurably affecting the constant weight of protein samples. When samples are dried a t 100" C., the associated trichloroacetic acid volatilizes completely, leaving residues identical in weight to those obtained when duplicate samples are precipitated by ethyl alcohol. Titration curves showed that, before drying, about 10% of the weight of an ethyl alcohol-washed trichloroacetic acid precipitate was associated trichloroacetic acid. After drying, trichloroacetic acid could not be detected by titration; and elemental analyses yielded gravimetrically insignificant amounts of organic chlorine. The gravimetric method was tested for precision by making replicate determinations of total protein, using l-ml. aliquots of six sera which had been diluted 100-fold with physiological saline. The standard deviation for individual samples was h 0 . 2 4 gram per 100 ml. of serum (11degrees of freedom), corresponding to 3.42y0 error because VOL. 31,
NO. 8, AUGUST
1959
1411
the proteins averaged 7.16 grams per 100 nil. of serum; this allows errors not to exceed 7.53% within 95% confidence limits or 10.62% within 99% confidence limits. Sitrogen in each welghed sample was determined by a Kjeldahl micromethod. For this purpose, the samples were dropped, crucible and all, into a borosilicate glass digestion tube measuring 25 mm. across by 150 mm. long. The samples were digested by boiling on an electric hot plate with 1.5 ml. of concentrated sulfuric acid and 100 mg. of a 1 to 3 copper sulfate-sodium sulfate mixture. -4ntibump rods. constructed by fusing a shallow glass cup to the end of a piece of glass rod, were used to ensure smooth boiling during digestion. When these rods are placed in a hot solution, the inverted cups yield a steady stream of vapor bubbles which
prevent superheating by agitating the liquid. The entire digest was transferred t o a micro-Kjeldahl apparatus, made alkaline, and distilled into a receiving vessel containing 5 ml. of 0.0100M hydrochloric acid, then back-titrated with sodium hydroxide, using methyl red as an indicator. The precision of the Kjeldahl micromethod was determined by analyzing replicate samples of a diluted serum. The mean and standard deviation for this series was 0.5306 + 0.0064 mg. of nit,rogen which allows errors not to
Table 111. Nitrogen Factors for Proteins in Human Serum
Determined using fractions separated by diethyl barbiturate buffer of 0.1 ionic strength at pH 8.6 using voltage gradient of 2.5 volts/cm. n
Table 1. Electrophoretic Mobilities in Agar of Proteins from Normal Human Serum
Determined in diethyl barbiturate buffer of 0.1 ionic strength at pH 8.6 using voltage gradient of 2.5 volts /em. n = 12 $3. . E -
Fraction Rho Albumin-1 Albumin-2 Alpha-1 hlphan-1 Alphap-2 Beta Gamma-1 Gamma-2 Gamma-3 Gamma-4 a Cm.2 volt sec.
Table 11.
of Meana Mean Std. Mobility Dev. (i)(*I 7.66 0.18 0.073 6.74 0.18 0.073 6.25 0.22 0,090 5.47 0.22 0.090 4.56 0.12 0.049 4.14 0.18 0.073 3.55 0 04 0.016 2.65 0.17 0.069 1.78 0.16 0.065 0.72 0 21 0.086 -0.22 -0 16 0 065
x 10-6
Fraction Rho Albumin-1 Albumin-2 Alpha-1 Beta Gamma-1 Gamma-2 Gamma-3 Gamma-4 Mean weighted factor for total protein
=
12
It Std. Mean Dev. Nitrogen for Factor" Individual 4.98 1.08 6,661 0.46 7.36j6,70b 1.46 5.90 0.69
6.13 7.117 5.80\6,60b 6.87 6.38J
0.86 2 08 0.27
0.73 1 12
6.60 wt. of protein a Xitrogen factor wt. of nitrogen * Pooled to show relationships to proteins separated by free-solution electrophoresis.
Distribution of Eleven Proteins Separated from Serum by Agar Electrophoresis
Determined in diethyl barbiturate buffer of 0.1 ionic strength a t pH 8.6 using voltage gradient of 2.5 volts/cm. ?i= 12 Mean % by f Std. Mean %;o of & Std. Fraction Gravimetry Dev., R Total Xitrogen Dev., % Rho 1.87 0.52 2.69 1.23 Albumin-1 5; :)'62 75a 6 23 55 291 6 30 -4lbumin-2 1 14 3 83j5' 1 01 Alpha- 1 6.14 3.19 5.44 1.30 Alpha2-l 4,05' 1.01 1.16 Alphar2 7,26)11.31" 1.24 3:f:1)11.69a 1.17 Beta 4.75 1 48 5.64 1.71 Gamma-1 2 31 0 55 1 18 Gamma-2 3 081 1 52 1 33 Gamma-3 6 22)13 1 73 1 65 Gamma-4 1 58) 0 84 2 07) 0 71 0
Pooled to show relationship to five fractions separated by free-solution electrophoresis.
14 12
ANALYTICAL CHEMISTRY
exceed 5.57% within 99% confidence limits. RESULTS AND DISCUSSION
Tn.elve healthy persons of Caucasian descent', six males and six females ranging in age between 27 and 47 years, donat'ed sera for these experiments. The c,oncentration of total protein in each serum was measured gmvimetrically by precipitating and weighing t,he proteins from 1-ml. aliquots of 1 to 100 dilutions of serum in physiologicnl saline solution. T h e n trichlorcacetic acid was used as the precipitant, the mean total protein was 7.26 0.52 (standard deviation) grams per 100 nil. j ethyl alcohol precipitation yielded a mean of 7.25 f 0.34 (standard deviation) grams per 100 ml. The mean nitrogen cont,ent of the precipitates 0.45 (standard deviation) was 15.15 yo corresponding to a nitrogen factor of 6.60. This factor did not vary with the precipitant. Elect'rophoresis of these sera resulted in separation of the proteins into eleven fractions. These fractions have been named to indicate their relationships to t,he fractions obtained by freesolution electrophoresis. Thus albumin-1 and albumin-2 are named to indicate t8he authors' belief that they originate in the albumin fraction of current, nomenclature. The fraction designated rho (for rapid] was reported and named by Williams and Grabar ( 5 ) . It is readily demonstrable by agar electrophoresis but not by other methods. Mobilities were determined for all fractions. When corrections were made for displacements due to electro-osmotic flow, the mobilities (Table I) agreed well with those reported for free-solution electrophoresis (4). The displacements due to electro-osmotic flow were determined by implanting agar strips with glucose, which is electrically inert, and measuring its displacement during the course of electrophoresis. This substance n-as chosen because its position can be determined easily by pressing a length of commercially available glucose t,est t'ape (Tes-Tape, Eli Lilly & Co.) against t.he agar surface. The n-eight of the fractions in t,he electrophorogmms from all subjects WBS det'ermined and used to calculate the mean percentages a.nd standard deviations shown in Table 11. The percentage distribution of Kjeldahl nitrogen, also shown in Table 11, was established by determining the nitrogen in the weighed precipitates. The marked differences between the percentage distribution of gravimetrically determined protein fractions and that of the associated nitrogen emphasize the danger of using a single nitrogen factor indiscriminately to estimate all serum proteins. Table I11 s h o m the
*
nitrogen factors for the individual fractions as well as factors lumped to conform with the five fractions obtained by free-solution or paper electrophoresis. While the factors for the composite fiactions are reasonably close t o one another, it is evident that very large errors would arise if a serum containing abnormal amounts of some subfraction Jvith a widely d i v e i g ~ n tnitrogen factor were analyzed. The large va.iations in nitrogen factors which are indicated by the standard deviations in Table I11 reflect the wide differences in the composition of corresponding fractions from different individuals. These variations arise in part from variations in the amount of nonpeptide material, such as lipides, associated with the migrating fractions. A much more important cause of variations is that the 11 fractions which are discussed are mixtures
rather than pure proteins, so that their nitrogen factors vary, depending on which component predominates. This complexity is easily demonstrated by excising fractions from a completed electrophorograrn, reimplanting them, and continuing electrophoresis. After runs totaling 80 em. in length, a t least 14 fractions can be distinguished. This technique can be used to extend the effective length of an electrophoretic migration; its usefulness is limited only because progressive attenuation eventually makes the protein precipitates impossible to see. Fractions in an untreated electrophorogram can be located by comparison with a second electrophorogram which has been run in parallel, then treated with trichloroacetic acid. There is additional evidence for the heterogeneity of electrophoretic fractions in the irnmunoelectrophoretic studies of Williams and
Grabar ( 5 ) , during which 16 or more antigens were found in human serum. LITERATURE CITED
(1) Gordon, A. H., Keil, B., Sebesta, I
