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KMID : 0383119550030040084
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Journal of Aerospace Medicine
1955 Volume.3 No. 4 p.84 ~ p.95
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Paper Electrophoretic and Paper Chromatographic Studies of the Blood Proteins. Part I : Studies on Paper Electrophoresis of the Serum Proteins
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Abstract
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In the first report (Theoretical Aspects and a Simplified Method of Filter Paper.
Electrophoresis of the Serum Proteins, ROKAF J. Av. Med., vol. 2.No, 2&3 pp.
18(1954)), the author considered some theoretical aspects of paper electrophoresis using
Kunkel´s glass plate method and found that the apparent mobilities of the seruni albumin are different due to the ionic strength of the buffer and in general, the separation into fractions was more apparent in high ionic strength unless the current denature the proteins. As a dye, the author selected eosin, because it takes only few seconds to stain. Filter paper also is a very important factor for the separation into fractions.
In the second report(Theoretical Considerations on Quantitative Estimation using Dye of Serum Protein Fractions Separated by Filter Paper Electrophoresis, ROKAF J. Av. Med., vol.3, No. 1, pp. 27(1955)), considered some factors in the quantitative estimation. Author´s eosin stainning method can not be applied for dye-elution method because eosin can not be eluated stoichiometrically after HgC12-alcohol fixation. Bromphenol blue was most suitable for elution method. Using Germany made electrodensitometer, the author checked the eosin stainning method, and found it also applicable for direct estimation on the paper without elution. In elution method, the electrophoretic curves were different with same serum on different papers. Accordingly, the selection of paper is very important from the stand point of quantitative estimation in addition to separatory capacity.
In the third report (Laboratory Make-up of a improved Electrophoresis apparatus and Electrodensitometer, ROKAF J. Av. Med., vol. 3, No. 4. pp. 76(1955)), the author devised a simple and improved electrophoresis apparatus including constant voltage rectifier(DC-output from 100 V to 450V) and a densitometer for direct measurement. In the case of amidbschwarz 10 B stainning, caesium phototube and 595 mμ filter must be used, and in eosin staining caesium-antimony phototube and 470 mμ filter should be used. In the densitometer, photocurrent can be easily amplified and its sensitivity is very high. Semi-automatic plotting device is also convenient for determination and some times oiling. process for transparency of paper can be omitted.
In this report, the author considered again buffer problems. In general, during phoresis, the electrical conductivity of buffer increases gradually, so the. intensity of current becomes high and thee voltage between both ends of paper becomes low. Barbital has a minimum such tendency. In low voltage below 100 V, ammonia buffer also utilizable. When potassium chloride-agar bridge is used, the concentration of potassium chloride is very important factor; because its electrical conductivity is low in under-saturated KCI-agar bridge. Accordingly, agar must be supersaturated with potassium chloride. From the theoretical standpoint constant voltage electrophoresis is superior than constant current electrophoresis but practically, in the case of high voltage, protein is liable to denature by Joule´s heat. But if less than 200 V is employed in room temperature, constant voltage phoresis is more favorable, because theoretical treatment is possible. The author found that apparent mobility of the rabbit serum albumin is slower than that of human serum albumin (rabbit serum albumin: 3.1x10-5 cm volt-1 sec-1 human serum albumin : 4.5X 10-5 cm volt-1 sec-1, in Wieland type phoresis using pH 8.6, μ=0.05-barbital buffer and Toyo No. 51 Filter paper.)
The author introduced two kinds of coefficient that is very useful for theoretical considerations. The one is specific mobility coefficient that is the distance moved by unit potential per unit area in unit time. The other is specific velocity coefficient that is the time needed to move unit distance by unit, potential per unit area. Using these coefficients and apparent mobility, one can calculate approximate time needed to move a certain distance of distance moved in a certain duration. In the calculation of the apparent mobility of albumin, the author found that10-3 times of ionic strength can be adopted as the approximate mean conductivity of buffer if calculated current from Ohm´s law is used.
Summarizing all results from the first report, the author standardized the method as follows.
1. Electrophoresis Apparatus:same as report 3.
2. Filter paper : Toyo No. 51, Whatmann No. 3MM, Schleicher-Schuel No. 2043 a&b.
3. Buffer.
a) Barbital buffer, pH 8.6, μ=0,1
In case of 200V, μ0.05
b) Ammonia buffer, pH 8.6, μ=0.1 (only low voltage use, below 100 V), Na CN added as much as 0,005%
4. Phoresis:
5. Staining: Amidoschwarz 10 B or Eosin.
6. Estimation: Direct densitometry.
Detailed data by the author´s standard method will be reported on next paper.
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