Optimal Fractionation Conditions for the Quantitative Analysis of Human Serum Protein Fractions by CelI ulose Acetate Electrophoresis COLIN J. BRACKENRIDGE Biochemisfry Deparfmenf, Royal Perfh Hospital, Perfh, Western Australia
b Several variables affecting the electrophoretic separation of human serum protein fractions on cellulose acetate have been studied and optimal conditions have been deduced. The choice of buffer, and the effects of ionic strength, pH and volume of buffer, length of membrane, position of setum application, voltage, and running time are shown to influence the over-all migration of the pattern and separation of protein bands. Based on the best conditions for staining and fractionation, a complete procedure has been devised for the quantitative analysis of five fractions to yield results in 5'/2 hours. The accuracy and precision of the method are satisfactory. Normal protein ranges, based on a sampling of the general population, have been determined.
T
HE CHOICE of opwating conditions governing the electromigration of serum proteins by membrane electrophoiesis is a matter 01importance since quantitatiw estimation depends on the sharp differentiation of fractions. Adverse conditions, such as low buffer p H values, cause coalescence of albumin and al-globulin ( 5 ) , arid exccssive membrane wetness blurs the pattern. The final separation is largely due to the nature of the buffer and the membrane. Thus nine fractions have been separated on cellulose acetate with the use of a special boric acid buffer; on paper incomplete frartionation resulted (1). In the present study, attention has been restricted to the five fractions of major clinical importance with a view to examining the factors which produce the most complete separation in the shortest time. The choice of variables is determined by the method of analysis; Tide bands and hence long running times are desirable for photometric scanning, whereas sharp bands and short patterns are suitable for elution of bound dye. Brief running times make the latter procedure more favorable for routine laboratory estimations and the relevant factors have been considered in this light. Thus in the equation defining the mobility of n migrant
d = - Ut!!
1
(1)
where d is the length of migration, u is the mobility (sq. cm. volt-' sec.-l), t is the running time, v is the voltage, and I is the effective length of the membrane, it is experimentally desirable to maximize d and minimize t. It follows that v must be maximized and I minimized. Maximizing of v is limited by the liberation of excessive heat with consequent evaporation from the membrane. Minimizing of I is subject to instrumental design and excessive wetting of the membrane. Optimal fractionating conditions, therefore. require a correct balance between these experimental extremes.
of Lissamine Green SF 150, 10.211 grams of potassium biphthalate, and 29.7 ml. of 1-V hydrochloric acid in water and make up to 1 liter. If necessary adjust pH to 2.7. Renew according to usage. WASHIKGSOLUTION.Make up 16 ml. of glacial acetic acid to 1 liter with water. ELUEXT.Dissolve 20.422 grams of potassium biphthalate and 91.6 ml. of 1N sodium hydroxide in water and make up to 2 liters, If necessary adjust pH to 6.0. The solution is stable if bacterial growth is suppressed with a thymol crystal. PROCEDURE
APPARATUS
The horizontal tank (Shandon Scientific Co. Ltd., London), specially designed for cellulose acetate electrophoresis, is a larger modification of that described by Kohn (4). It is capable of holding four 18 x 5 cm. strips or eight 18 X 2.5 cm. Strips. and has internal dimensions of 23.5 X 21.5 X 5 em. The buffer compartments of total capacity, 1300-nil.. occupy the whole area of the tank, thus providing a continuous fluid surface. Two bridges, across which the strips are stretched, extend over the width of the tank and are shaped as quadrants of a cylinder. Three notches into which the bridges fit allow the exposure of a variable length of strip. Half-width strip holders shaped to the curvature of the bridge keep the membrane in position under moderate tension. Filter paper is interposed between strip and holder t o provide a connection between strip and buffer. The tip of a micropipet, calibrated to deliver 10 pl., was finely ground to an external diameter of 1 mm. to facilitate addition of serum. REAGENTS
BUFFER.Dissolve 1.96 grams of anhydrous sodium acetate, 5.00 grams of sodium diethylbarbiturate, 3.42 ml. of IA' hydrochloric acid, and 10 ml. of glycerol in water and make u p to 1 liter. If necessary adjust p H to 8.6. Renew weekly. DENATURANT. Dissolve 30 grams of sulfosalicylic acid in 1 liter of water. Renew every 2 weeks. DYESOLUTIOS. Dissolve 0.500 gram
Cellulose acetate strips (18 X 5 cm.) are prepared by overnight immersion i ti buffer solution. Place four strips, free of surfacv buffer film, in position in the tanh. Apply 10 111. of serum as a line 2.5 cm. from the center on the cathodic sidr. Adjust the potential to approximately 160 volts. One hundred minutes late], remove each of the strips and immerse in the denaturant for 20 minutes. Poui off the solution, wash strips twice with water, remove surface moisture from each strip, and immerse in a dye bath maintained a t 30" C. After staining for 135 minutes, remove the strips and place them in the washing solution. Agitate until no more excess dye colors the solution; three rinses are usually sufficient. Remove the strips, and as the surface moisture evaporates cut out the five fractions. Place the segments into large tubes containing measured volumes of eluent. Optimal volumes depend on the amount of protein-bound dye and characteristics of the spectrophotometer. Shakc and mix well a t intervals until elution is complete (usually within 30 minutes) ; the solution is stable for several clays at room temperature. Measure the absorbance of each eluate solution and calculate the weight of dye absorbed by each fraction from a calibration chart relating dye concentration t o ahsorbance. The concentration of each protein fraction in grams per 100 ml. of serum is then estimated from the dye, uptakes listed in Table I. With this procedure, results are obtained about 5'/2 hours after applying the serum. Each strip containing 10 pl. of normal human serum takes up VOL. 32, NO. 10, SEPTEMBER 1960
1357
I.
Dye Uptakes of Human Serum Protein Fractions
Table
Grams Protein/ 100 M1. Serum 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Aa
Dye Bound by a 6 G
9 24
5 12
40 _.
22
56 72 88 104 120
~~
32 42 52 63 74
1 12 20 28 36 14 54 63
4 14 24 35 46 56 67 78
153 164 174 185 196 207
233 250 266 282 298 314 33 1 347 363 379 396
4 Symbols as for Table I (p. 1353, this volume) (8).
Table II. Accuracy and Precision of Method Mean & S.1). in Grams/ 100 hI1. Serum" ____-__
ElectroBiuret Protein phoresis CuSO, Albumin 3 80 f 0 08 3 76 f 01.21 a]-Globulin 0 27 f 0 03 or2-Glohulin 0 61 & 0 04 @-Globulin 0 77 f 0 05 */-Globulin 1 13 =k 0 08 Total globulins 2 7 8 3 ~ 014 2 8 4 f 0 .16 Total proteins 6 57 & 0 13 6 60 i 0 .30 Rased on 24 determinations.
approuimately 0.5 mg. of dye from the bath. The dye-bath should be renewed p(4odieally with this in mind. STUDY
OF
VARIABLES
AND
DISCUSSION
Choice of Buffer. Tris(hydroxyniethy1)aminomethane (tris buffer) has been recommended for use in electrophoretic buffers (8), and is claimed to possess definite advantages over the usual veronal mixture because of its greater buffering capacity and loil-er molar ionic stiength. When compared with the modified Michaelis buffer used by Owen ( 7 ) , the tiis-veronal buffer mixture did not yield as long a migration distance so further study was not pursued with it. Except where otherwise mentioned, a sodium acetatesodium diethylbarbiturate-hydrochloric acid mixture was adopted. Effect of Ionic Strength of Buffer. Buffer solutions of ionic strength
1358
ANALYTICAL CHEMISTRY
0.03, 0.06, 0.09, and 0.12 were prepared by altering t h e sodium acetate content. and t h e senaration of fractions an'd t h e total lehgth of migration were noted in each case. Under conditions of p H 8.66, %hour running time, and 2 ma. per strip, maximum migration was achieved at 0.05 ionic strength. This was adopted as the optimal value. At values lower than this, there is an increased separation of globulins and a decrease in the albumin-al-globulin distance. At values above 0.05, the reverse effects take place, and the result of increasing the running time is only to lengthen the alhumin-a1globulin distance. Effect of pH of Buffer. \Then the buffer p H is varied between 7 and 9 at a constant ionic strength of 0.05, no change in over-all migration lrngth is observed, b u t there is t h e expected decreased migration of albumin a t lower p H values as the isorlectric point is approached. Concomitantly the albumin-cui-globulin separation is reduced and t h e distance betn-een the 0-and 7-globulins increases. Since the p H in the original reference was 8.66 (7) , this figure was chosen as suitable. Effect of Buffer Volume. The rffertive length of strip and t h e volume of buffer both control the wetness of the cellulose acetate. As the buffer volume is increased from 500 to 800 ml., there is a slight decrease in the migration of albumin while t h c overall distance remains approximately the same. h broadening of t h e albumin band with consequent engulfing of al-globulin is also noted. Because of this, and for reasons of rconomy. 500 ml. was selected as the best volumr. Effect of Length of Strip. When the length of strip between buffel surfaces is fixed a t 10.4, 12.4, and 14.4 cm., the over-all length of niigration is significantly shorter a t 14.4 em. t h a n a t the two lower dista~icos where the observed length is t h r sanir. There appears to be a progressive broadening of fractions a s the effective length of strip is diminished. I n choosing 12.4 cm. as optimal, a compro-
Table 111.
ACCURACY A N D PRECISION
The accuracy of the metliod was checked against the results obtained on a pooled human serum with a hiuret copper sulfate method (IO) modified by the use of 2G.8% sodium sulfate as the total protein precipitant ( 6 ) , and using standards based on the Kjeldahl nitrogen content. The reliability of the biuret mvthotl has l w n confirmed
Normal Ranges of Human Serum Protein Fractions"
Protein
Concen t,ratioii Mean f 2 S.11.b
illhumin a,-Globulin a2-Globulin @-Globulin -{-Globulin Total globulins Total proteins
4.24 f0.52 0.28 f 0.06 0.74 & 0.17 0 . 8 8 =t0 . 1 5 1.26 f 0 . 3 1 3.16 & 0 . 3 8 7.40 & 0 . 6 9
a
mise has been made between extreme effects. Effect of Position of Serum A p ~ l i cation. As the serum is added^&ther towards t h e center of t h e effective length of strip, the migration distance of albumin increases. At t h e same time t h e total separation is enhanced and the albumin-al-globulin separation improves. It is advisable to choose the position of application so that i t coincides with the 7-globulin band; in this way nonmigrating material such as macroglobulins cannot obscure the other fractions. Addition at a point 2.5 cm. on the cathodic side of center produced the best results. Effect of Voltage. The distance travelled by the migrant should he directly proportional to the voltage according to Equation I , and this is observed under the present conditions. As t h e wattage increases. t h e albumin migration drops, and t h e over-all length increases. The albumin band broadens yet t h e separation from a,-globulin grows appreciably as the remaining globulins migrate in the opposite direction. There is also a splitting of 0-globulin. Three and two tenths volts per mi. of effective length of strip and O.G nia. per cm. width of strip yieldrd optimal fractionation. Effect of Time. T h e a r i t y of funetion was observed between running time and albu~~iiri-~-globulin distance. At longer time iritc~rvalsthe albuminal-globulin vparatioii markedly increased anti all protcin bands became vague and diffusr. 0111, hundred minutes iva? found to I ) t h a suitable time.
Comparison of 5 Total Prot'ein Cellulose Paper acetate (3 5i.3 3.8 10.0 11.9 17.0 42.7 100 0
Frequency Distribution
59.2 4.1
8.4 11.4 16.6 40.5 99 7
Based on presumably normal sprn from 20 males and 20 frm:~les;mc:m 100 ml. serum.
* Grams per
Normal Left skenRight sken Xormal Normal Left skemNormal ngc 35.
t)y analyses of standard protein solutions and standard control sera. Table I1 lists the means and standard deviations of various protein concentrations estimated by both procedures based on 24 determinations. The agreement for the concentrations of albumin, total globulins, and total proteins is very satisfactory. The electrophoretic method yielded a higher degree of precision than the chemical fractionation procedure. N O R M A L RANGES
Serum was collected from 40 presumably healthy persons, 20 of each sex, in order to detcrmine the normal i'anges of electrophoretic protein fractions. ,4n attempt ivas made to base the selection on the agc distribution of the general population; the average age was 35, S o two persons came from one family. Table I11 summarizes the data ohtained. The ranges were calculated to include 95aJ, of the persons chosen. The albumin-total globulin ratio, frequently used as a standard for protein halance, yielded a mean i. 2 standard (leviation of 1.34 f 0.19. Comparison of '1'ahl(~s I1 and 111
revCali: that the standard deviations of the normal ranges vary only u p to three times those of the precision of the method. This is difficult to reconcile with the finding of Strickland et al. (9) t h a t corresponding protein fractions from different sera varied widely in their abilities to bind the same dyes. They concluded t h a t large errors can result from dependence on this method which can be expected to detect only gross ahnornialities in serum protein distribution. Lissaminc Green was not one of the dyes studied. Ehrmantraut ( 3 ) has calculated tht. mean normal values of serum protein fractions, expressed as percentages of thc total, obtained from the normal values from 36 published studies on paper electrophoresis. When compared with those det,ermined in the present investigation, the agreement is good (Table 111),the largest difference being in the a2-glohulin fraction. Histograms werc conptructed to determine the frequency distriliution of the normal values of each fraction; three appeared to be normal and two were skewcd. Since electrophoretic fractions coristitute the 511111~of many suhfractions. thcir signi-
ficancr 1s difficult to ai.es+.. X o significant differences between sexes were found, but after completing over 200 analyses covering all age groups, a tendency has been noted for the albumin to fall and both a2-globulin and yglobulin to rise with increasing age. LITERATURE CITED
(1) Aron~son,T., Gronwall, -4., Scand. J Lab. (Tin. Znuest. 9, 338 (1957). ( 2 ) Brackenridee. C. J.. h 4 1 , . C:HEM. 32,
-
1353 (1960). (3) Ehrmantraut, Ei. C., "The Clinical Significance of Paper Electrophoresis," ~
Fipinco Division, Beckman Instruments, Inc.. Palo Alto. Calif., 1958. . .4cta 3 * 450 , , (1958).
( 5 ) Laurell, C. B., Laurrll, S.,Skoog, S . , Clin. Chern. 2 , 99 (1956). (6) Milnr, J., J . Bid. Chem. 169, 5% ( 1947). ( i )Owen, J. A,, Analyst 81, 36 (1956). (8) Rapp, K. I]., Memminger, 11. AI., A n i . J . Clin. Path. 31, 400 (19,59). (9) Strickland, R. I)., Podleskl, T. H.,
Gurule, F. T., Freeman, 31. L., Childs, JV. A , , ASAL. CHEJI.31, 1108 (1950). (10) JVolfson, IT7, Q.) Cohn, C., Calvary, I,:., Ichiba. F., .1 T U . J . ('!in. Path. 18,
Variable Dye Uptake in the Quantitative Analysis of Abnormal GIo b uI ins by Cel I uI ose Acetate EJectrophoresis I
COLIN J. BRACKENRIDGE Biochemistry Department, Royal Perth Hospital, Perth, Western Australia
b Examination of 28 serum samples containing abnormal globulins has shown that paraprotein dye uptake is variable owing to instabiliiy. Since prediction of dye-binding behavior seems impossible, the total protein content should b e determined by an independent method to arrive a t the concentration of the abnormal component.
I
THE previoudy described procedure for the quantitative estimation of human serum protein fractions if ), it has been assumc3d that no irregulaiitg in dye uptake occurs other than small contributions by differing amounts of individual proteins within 3. given electrophoretic fraction. The observation t h a t the standard deviations of the normal physiological ranges of any of the fractions never exceeded t h t w times thow of thr. pi~cision of
x
thv method supports this claim. ?'her,r is, howver, the possibility that on rare occasions in normal persons, or in disease states, conditions might arise to invalidat'e the assumption. Physicochemical instability, molecular aggregation, or protein-protein interaction cannot be discounted from sometimes occurring in vitro or as a methodologicaal artifact. The present investigation deals with the dye-binding properties of the socalled serum paraproteins which are abiiornial in a quantitative or qualitative sense depending on whether they are formed by the selective increase of a particular protein component or as a different species from those present in normal and most pathological sera. '1'0 this class belong the cryoglobulins, macroglobulins, myeloma proteins, and aa-glohulins ( 5 ) . The latter are unstable prot'eins 1%-hichmigrate b e t w e n the a?- and 8-globulins! while t h r othcre
have mobilities of CY?-, 6-, or y-globulins or intermediate fractions such as AI-type paraproteins which migrat'c between 6and y-globulins. Although sevwal quantitative electrophoretic studies of such proteins have been published (a, 3 ) , scant attention has been paid to their d p b i n d i n g capacities. The work to be drscribcd arose from the question of whether their dye uptake was regular 01' variable, and, if the former, to which fraction they bore the most similarity. PROCEDURE
Sera of patients suffering from essential cryoglobulinemia, maci oglobulinemia, and myeloma are subjected to the quantitative electrophoretic procedures already described ( 1 ) and the protcin pattern is inspected. I n most cases a discrete abnormal band is visible in t h r g l o h l i n region. If it is superimposed on one of the fractions, the segments arc out out and thc dye is eluted as usual. VOL. 32, NO. 10, SEPTEMBER 1960
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