In the Laboratory
Quantitative Protein Determination from Cellulose Acetate Strip Electrophoresis M. Barufaldi, N. B. Pappano, and N. B. Debattista* Department of Chemistry, San Luis National University, Chacabuco 917, 5700, San Luis, Argentina; *
[email protected] A laboratory practical work has been designed for the quantitative determination of serum proteins by means of rapid vertical electrophoresis, using standard curves. This practical work is aimed at students in the biological fields to help them become familiar with diverse methodologies used in clinical analysis. Theory Electrophoresis, defined as ion migration under the influence of an applied electrical field, is a technique for the separation of macromolecules as a function of their charge and size. Charged species in an electrical field, E, move toward the oppositely charged electrode (1). The electrophoretic mobility of a given particle depends in part on its total charge at a given pH and ionic strength. The strength acting upon the charged solute molecule is given by e z E, where e is the electronic charge and z the number of charges on the molecule. Each ion is accelerated for a very short period of time, then reaches a uniform rate, since the electrostatic strength is balanced by the frictional force exerted by the solvent medium. At this point, the ions move at a constant rate, v. Thus, ez E = f v = 6πη rv
(1)
where f is the friction coefficient of a spherical particle (2) (Stokes’ law), η is the viscosity coefficient, and r the particle radius. It can therefore be stated that
v = e zE 6πηr
(2)
The electrophoretic mobility, µ , is defined as the rate per unit electric field
µ = v = ez E 6πηr
(3)
with units of m2 V {1 s {1. This is a simplified expression that does not take into account the influence of ionic atmosphere on the movement of the ions. Proteins are macromolecules that typically contain groups that may carry a charge (3). The net charge of proteins varies with solution pH. Using a buffer solution with a pH higher than the isoelectric points of the proteins to be separated allows separation of the component proteins. Since the mobility of protein fractions depends on size and electric charge, albumins move at a faster rate than γ-globulins.
Experimental Procedure
Equipment and Material Electrophoresis Mini-Vertical Gel Systems Models EC185-A 6412 (EC-Apparatus Corporation), of low exposure time Power Source EC 135 supplying pure continuous current CAMSPEC UV–vis digital spectrophotometer Strips of gelatinized cellulose acetate (2.5 cm wide and 17 cm long) preserved in 50% methyl alcohol (Chemetron Milano) Veronal–sodium veronal (Sigma Chemical Company) buffer solution, pH 8.6 and ionic strength 0.05 M Staining solution: Amidoblack 10B (0.6%), acetic acid– distilled water–methanol 10:45:45 (Sigma Chemical Co.) Destaining solution: acetic acid–distilled water–methanol 10:45:45 Eluent: sodium carbonate (3%, Sigma Chemical Co.), distilled water–methanol 50:50 Bovine serum albumin, V fraction (99%) (Sigma Chemical Co.) Human serum (pH = 7.2); CAUTION: The human serum samples must first be analyzed in a clinical laboratory.
Procedure The cellulose acetate strips were submerged for 10 minutes in the buffer (4). When they were taken out, the excess liquid was removed by placing the strips between two pieces of filter paper. Buffer solution was poured in both compartments of the electrophoresis apparatus. The cellulose acetate strips were placed on the glass support with the most opaque side up. Sample application was carried out with a micropipet. The electrophoresis unit was connected to the continuous current source for 30 minutes (200 mA). After that time, the strips were removed and submerged in the staining solution for 5 minutes, after which they were destained. The strips were cut to separate fractions, which were placed in separate test tubes containing 10 mL of the eluent for albumin and 3 mL for other fractions. The fractions were kept in the tubes for 10 minutes. A control was prepared with 10 mL of eluent and a piece of strip the same size as that of the destained fractions. A calibration curve was prepared using bovine serum albumin (BSA). A standard BSA solution was prepared in distilled water (0.9% w/v). The pH was adjusted to 7.0 with 0.1 N sodium hydroxide. Different volumes of the standard solution were applied to each strip and electrophoretic runs were made according to the procedure described above.
JChemEd.chem.wisc.edu • Vol. 76 No. 7 July 1999 • Journal of Chemical Education
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In the Laboratory
Results and Discussion
Table 1. Electrophoresis of Bovine Serum Albumin at 20 ºC Application Vol/ µL
Protein/ µg mL{1
Absorbance λ = 620 nm
5.0
4.50
0.133
8.1
7.60
0.230
9.00
0.269
10 13
11.4
0.330
15
13.5
0.376
17
15.2
0.410
20
18.0
0.432
Table 1 shows the data used to create the calibration curve for bovine serum albumin. Absorbances were read at 620 nm. Figure 1 shows the calibration curve for bovine serum albumin. Using the described procedure, the protein fractions of human serum samples were quantitatively determined. Using the BSA calibration curve, the protein concentration in µg/mL was obtained. By the following calculation
C (g%) =
N OTE: Data represent the mean values from 5 trials.
Table 2. Human Serum Analysis Absorbance λ = 620 nm
Concentration/ g d L {1
%
Albumin
0.220
3.65
55.9
α1-Globulin
0.090
0.45
Protein Fraction
6.90
α2-Globulin
0.120
0.60
β-Globulin
0.150
0.75
11.5
γ-Globulin
0.220
1.08
16.5
N OTE: Application volume is 2 ×
10 {3
9.20
mL.
protein mass × dilution application volume × 1 × 106
× 100
the percentages (w/v) of different protein fractions are calculated. In quantitative determination of protein fractions by traditional methods (4), the absorbances are summed and the corresponding percent values of the fractions are calculated. Knowing the total protein value (Lowry micro method, kit and reagents Sigma Chemical Co.) and the individual percentage values, the protein concentration in each fraction (g/100 mL) can be obtained (5). Our innovation is the use of the calibration curve to determine the concentrations of protein fractions separated by electrophoresis. Table 2 shows data obtained from human serum samples by this procedure. Conclusions This three-hour laboratory procedure introduces students to a routine technique used by clinical analysis professionals. The results obtained by this method are as accurate as those obtained by traditional ones, and our method has the advantage of greater simplicity and reduced time. Aknowledgment We wish to thank for support given by San Luis National University, Argentina. Literature Cited
Figure 1. Calibration curve for bovine serum albumin.
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1. Van Holde, K. E. Physical Chemistry; Prentice Hall: Englewood Cliffs, NJ, 1971; pp 123–130. 2. Atkins, P. W. Physical Chemistry, 5th ed.; Oxford University Press: Oxford, 1994; p 795. 3. McMurry, J. Química Orgánica; Grupo Editorial Iberoamérica: México, 1994; pp 1038–1048. 4. Ióvine, E.; Selva, A. A. El Laboratorio en la Clínica, 3rd ed.; Médica Panamericana: Buenos Aires, 1985; pp 238–239. 5. Brey, W. S. Physical Chemistry and its Biological Applications; Academic: New York, 1978; pp 469–471.
Journal of Chemical Education • Vol. 76 No. 7 July 1999 • JChemEd.chem.wisc.edu
