Optimal Staining Conditions for the Quantitative Analysis of Human Serum Protein Fractions by Cellulose Acetate Electrophoresis COLIN J. BRACKENRIDGE Biochemistry Department, Royal Perth Hospital, Perth, Western Australia
b As a basis for the quantitative electrophoretic analysis of five major human serum protein fractions, the optimal conditions for staining with Lissamine Green on cellulose acetate have been elucidated. The effects of pH on dye elution, and of pH, ionic strength, staining time, and staining temperature on dye uptake are examined and applied to the measurement of dye-binding capacities of five plasma proteins, one mucoprotein, and two lipoprotein preparations. Optimal dye uptake occurs at a pH related to the apparent dissociation constant of Lissamine Green. Calculations on the kinetics of staining indicate that the optimal staining time chosen is appreciably longer than the half life of the reaction. Staining should be thermostatically controlled because of the relatively high temperature coefficient of albumin. The dye uptakes of albumin and four globulin fractions were virtually linear with respect to concentration, and confirm the suitability of Lissamine Green for staining proteins.
S
INCL the introduction of paper as it medium for zone electrophoresis,
other materials have been proposed from tiinr to time to improve the fractionation of complex mixtures. Kohn ha< described the properties of cellulose acetate and found the membrane sriitablr for immunodiffusion technique-, inimunoelectrophoresi? and mic.roelectrophoresis (6, 11). Smith and Xurchison (12) have descrihd ieveral of it3 advantageb over filter paper in the electrophoretir separation of strum protein fractions. Only microliter amounts of serum m required and no spreading occurs on application. Sharp separation of fraction?, nith no tailing of hands, 1- a?compli4icd n ithin 2 hours and a rampletclr white background i. obtained after \taining. For quantitation by photometric scanning, coinpletp transparencv in the clearing fluid minimize< errorcause of dye instability at, highcr p1-I the value of (j.0 \vas chosen; :tblr alberriativc ~ o u l dbe bet\vevn pH 0 to l . The minimal quantit,j. I s l u t c d a t ]JH2.9 appcsarcd t o indicate, that iiusimal dye ul)t:il;c, \vrJ1ild O C C U ~ :it this v:ilut.. Effect of pH on Dye Uptake. b~yual amounts of the sanie seruni \vert' applied to :I crllulosc acetatcl stril). K h r n dry, thca protc,in spots were denaturrd m d p l u c ~ d in Lissnmiiic Green solutions of id(bntica1
110
-
w >
-
1
/?
'
* ELUENT
ABSORBANCE
pH
Figure 2. Effect of pH on elution of protein-bound Lissamine Green The ordinate scale measures both the weight in milligrams of eluted dye and the absorbance at 635 mM
concentration but varying pH. Potassium biphthalate-hydrochloric acid buffers were used. Following vlution in p H 6.0 buffer, maximal (lye uptaktl had taken place a t about I I H 2 . i (Figure 3), close to the figure obtained in the previous experiment. It would therefore appear that optimal dairiing occurs at the pH at which ionic :ind undissociat'ed forms of Lissamine Green exist in equal proportions. To test the general validit!. of this result, the experiment wax repeated on filter paper and with albumin and 7-globulin. . h i acetate buffer was also used. Thc present figure is iridcpendent of protein fraction or supporting nicdium but the shape of the curve is influenced by the ionic strength of the buffer solution. 'l'hc clilut,t, acetic. avid washing wlution
0
- -
PH
Figure 3. Effect of pH of dye bath'on the uptoke1in milligrams of Lissamine Green The weight of serum protein was 330 y
1354
ANALYTICAL CHEMISTRY
00
-
70
-
0.1
0.2
0.3 IONICSTRENGTH 0.7
0.8
0.9
Figure 4. Variation of Lissamine Green uptake with ionic strength of dye bath. The weight of serum protein was 130 y
P
I
I
0 7
I
04
I
0.3
I
1 /
0.4
yGLOBULIN
levels are approximately 4 g r a m :mi 1 gram per 100 ml., respectively, :t time was chosen close to the intersection of the curve5 draMn at about these concentrations. The optimal itaining time selcrted \\as 2.25 houri. This ensured that thc period of rqiidly changing dye uptake \\as exceeded and t h a t the approximation to equilibrium was reasonably caloie. The per cent completion of the \tnining process a t variouS protein fraction concentrations can hr ralculatcd from the reaction kinetics. .\ssumiiig that the staining rate varies n i t h .om(. pon er of the protein conrentrB t'I o n : IDldT
, 25
50
MINUTES
125
150
Figure 5. Variation of the dye-protein ratio (D/P) with staining lime Grams per 1 0 0 MI. Albumin 4.84 2.42
9
0.97
0
0
y-Globulin 4.34 2.17 0.87
should have a pH of about 2.8. Accordingly, a 1.67, v./v. aqueous solution was chosen as thc optimal concentration. Effect of Ionic Strength on Dye Uptake. Equal amounts of the same serum werc applied t o cellulosc acet a t e st,rips. When dry, the protein spots were denatured and placed in 0.0,i70 w./v. Liss:iminc Green solutions of pH 2.7 and ionic strengths varying from 0.1 to 1.0. These rverr prepared by the :tddition of the calculat,ed weight of sodium chloride. Figure 4 indicates that the dye uptake, calculated from tlw absorbanw of the pH 6.0 (llu:it,e, is approsimatc~l!- Ixilved over tlic range of ionic strengths qtudied. Effect of Staining Time on Dye Uptake. It is dc>sirablr t h a t the variation of tiye uptake 15-it11 protein conccmtration be linear, a n d this will be dq)c>n&wt on thc staining tinic. Thc opt'inial staining t'iine may be deteriiiined as t h e point of iutt.rscction of plots of thc dye-probein ratios at particular protein concentrations z's. time ( 2 ) . The dye-protein ratio (D/P) is t'hc weight of dye taken up I i w gram of protein. Using the conditions and procedure :tlreacty described, curves d r a \ w b\p1ott)ing the dye-protein ratins at three concentrations of albumin and yglobulin against the staining time in minutcs are shown in Figure 5 . In neither case is there a single point of intersect,ion, indicating that linearity of dye uptakc is unobtainable over the conwntration range studied under the couditions used. Sinw the normal human serum albumin :ind y-glohulin
=
t
40
i t
35
30
/
-25
'G +io
2c 15
10
5
IzPn MINUTES
where D 1s thc n right of dye ill in1111grams takrn up by P milligrams of protein in T minute., and 72 is a proportionality cwnstant, under conditions of linear dye uptakc P may be rrlated to (S-D) where S' is the saturation weight of dye. The value of the power n was ~ a l i i a t r d liy conventional methods a i h o . T h u h integration of the qecond order equation
Figure 6. Variation of the time-dye uptake ratio (T/D) with staining time Numerols refer to concentrations in grams per 100 ml. of serum albumin (A) and y-globulin (GI
7-
400 350
-
300
-
W
>.
n ? - -
Figure 6 demonstrates the applicability of this e q u a h n t o describe the kinet>ics of staining; linear plots of ( T i D ) zw. T in minutes are obtained over large concentration ranges of albumin and y-globulin solution*. Table I1 summarizes the values of dyc~ saturation level, reaction constant, and per cent completion of reaction in 2.25 hours. Experimental conditions n w e those described prwiousiy. 10-p1. amounts of protrin solution being staincd a.t 30' C. Effect of Staining Temperature on Dye Uptake. If t h e staining process is appreciably temperature-dependent,
Table II.
150
-
100
-
10
I )il~itiori~
.41 .-i2
A5
G1 G2 cr5 0
4.98 2.49 1.00 4.23 2.12 0.85
15
20
25
30
DEGREES C.
Figure 7. Variation of Lissamine Green uptake with staining temperature Numbers refer to concentrations in grams per 100 ml. of serum albiimin (A) and y-globulin (GI
Kinetics of Protein Staining at
Protein
Concn., Cr./100 111.
-OG+
n
0
s, hlg. 0,500 0.208 0,065
0.257 0.111 0.037
li Mg.
x
30" C. 10,
Min. -l
%;o c'om-
0.501 3.06 i'ery high 1.61 12.3 Very high
pletion
-I i
90 100 87 96 100
Albumin (A) and y-globulin ( C ) dilutions corresponding to tho second column ;
see Figure 6.
VOL. 32, NO. 10, SEPTEMBER 1960
1355
250
I
7-
/ i
I
A
/
/
/
200
G
/
>
300
,/
2 25 50 0
G/
w
35011
/'
w
0
&
is
A
100
150 100
50
50 0.5
1.0
20 2.5 3.0 grams PROTElNjlOOml 1.5
3.5
4.0
1
Figure 8. Uptakes of Lissamine Green by eight serum protein fractions Captions refer to albumin (A), fibrinogen (F), 81-metal-combining globulin (BIM-CG), y-globulin (G), az-mucoprotein IanMP), pa-globulin (&GI, al-lipoprotein (allP), and &-lipoprotein (P,LP)
variations of room tempeiatuie may be expected t o introduce errors in dye uptake measurements. Serial dilutions of serum albumin and y-globulin covering the normal physiological ranges were stained over the temperature range IO" to 30" C. Figure 7 shows t h a t the temperature coefficient of 7-globulin IS negligibly small, b u t that of albumin IS significant a t all concentrations, especially the higher. Thus at a level of 5 grams per 100 ml., the uptake is 14% higher at 30" C . than at 10' C. This makes thermostatic control of the staining process essential and 30" C. was chosen as reference temperaturt. Dye Binding of Protein Fractions. Using t h e procedure a n d optimal experimental conditions described, the variation of dye uptake with concentration of eight protein fractions is shown in Figure 8. Linearity ocrurs ovei most of t h e ranges. All globulins have a lower uptake t h a n albumin or fibrinogen. The two lipoprotein samples have identical uptakes, so also do a?-mucoprotein and &-globulin. The five main fractions of clinical importance are albumin, and al-, a2-, p-, and y-globulins. The uptakes of the pure intermediate globulin fractions were calculated from the data in Table I and Figure 8 by solving three simultaneous equations. It is assumed that the dye uptake of the polypeptide moiety of a particular conjugated protein does not vary within t h a t electrophoretic fraction. The dye-binding capacities of pure albumin and yglobulin determined in Figure 8 were used in estimating their contribution as impurities to the true uptakes of al-lipoprotein, a%-mucoprotein, and @?-globulin. The contribution of fibrinogen to the final column of percentage composition in Table I ww ignored in view of its low concentration in plasma. When the uptakes in micro1356
*
ANALYTICAL CHEMISTRY
011
-C 0.16
012
Captions refer to albumin (A), yglobulin (GI, a-globulin (a),and globulin (B)
+ 0.146 8.1, 21.8, 58.5, 97.5
+ 0.81 + 0.10 p
U.28
01.
13.8, 34.3, 82.5, 131.5 0.01
cri
5
Figure 9. Corrected uptakes of Lissamine Green by major protein fractions
grams due to albumin and 7-globulin were subtracted from those of the impure al-, a2-, and p- fractions at concentrations of 0.5, 1.0, 2.0, and 3.0 grams per 100 ml., the equations to be solved were : 0.61
2 3 4 grams PROTEIN/lOOml.
+ 0.04 + 0.86 6 =
p-
enhances its suitability as a protein stain. ACKNOWLEDGMENT
The author gratefully acknowledges the gift of five globulin samples from the Commonwealth Serum Laboratories, Parkville, Victoria. This work was undertaken a t the suggestion of D. H. Curnow to whom appreciation i:, expressed for his guidance and interest.
IY?
15.0, 33.5, 77.5, 123.6
After solution of the equations, the uptakes of the pure fractions were almost linear with respect to their concentrations (Figure 9). Those of the al- and a?- fractions were identical within experimental error. Klotz has deduced a relation between the dye uptake and the maximum number of protein-bound moleculev by treating the protein molecule as a series of independent ions (9). It was shown that one molecule of bovine serum albumin could bind a maximum of 22 methyl orange anions (IO), while Franglen (6) calculated that the maximum uptake of bromocresol green is 24 moles per mole of human serum albumin. Taking the molecular weight of human serum albumin as 69,000, a maximum uptake of 68 moles of Lissamine Green per mole of albumin was obtained from the data of Figure 9. Since methyl orange and bromocresol green each contain one sulfonic acid group and Lissamine Green contains three, it appears that the dye uptake is dependent upon the number of acidic groups present in the molecule, and that the albumin molecule is able t o bind u p to about 23 times the number of acid groups in each dye molecule. Thus the increased number of sulfonic acid groups in Lissamine Green further
LITERATURE CITED
(1) Blomback. H., .ii-k. Kerrii. 12, 99 (1958). (2) Brackenridge, C. J., J . CXn. Path. 13, 149 (1960). (3) Brackenridge, C. J., ANAL.CHEM.32, 1357 (1960). (4) Color Index, 2nd ed., Vol. 3, p. 3351,
Chorlev and Pickersaill, - . Ltd.. Leeds, 1956.
( 5 ) Consden, H., Kohn, J., Nature 183, _1.512 _ ~ _(1959'3. -
(6) FraAglen,' G., "Paper Electrophoresis"
G. E. W. Wolstenholme and E. C. P Millar, eds., Ciba Foundation Symposium, p. 172, J. %I A. Churchill Ltd.. London, 1956. 17) Gorrinee. J. A . L.. Clin. Chini. Actu 2, 353 (1%7). (8) Jacobs, S., n'ature 183, 1326 (1959). (9) Klotz, I. >I,, rlrch. Biochun. 9, 109 (1946). (10) Klotz, I. AI., Walker, F. M.,Pivan, R. B., J . 4 7 ~ .Chem. Sot. 68, 1480 (1946). (11) Kohn, J., .lraturs 181, 839 (1958). (12) Smith, U. C., >Iurchison, JV., J . M e d . Lab. Tech. 16, 197 (1959). 113) St,rickland, R. D., Podleski, T. H., Gurule, F. T., Freeman, 31. L., Childs, W.A,, ANAL.CHEW31, 1408 (1959). (14) Sunderman, F. W,, Jr., Sunderman. F. W.,Clin. Chem. 5 , 171 (1959). 115) Suiiderman, F. W., Jr., Sunderman, F. IT., Falvo, E. A., Kallick, C. J., Am. J . Clin. Path. 30, 112 (1958). (16) Yeoman, W. B.. Clin Ch??ii. .Ida 4,523 (1959). 1IEcEIvrw for review March ?5, I960
Accepted June 23, 1960
