R. A. BROWN,J. P,. SIIlTMAKER,
4420
[CONTRIBUTION No. 1539
FROM THE
JR.,
J. R.C A N N AND J. C,. KIRKWOOD
Vol. 73
GATESA N D CRELLINLABORATORIES OF CHEMISTRY,CALIFORNIA INSTITUTEOF TECHNOLOGY ]
Experimental Investigation of Conditions of Transport in the Electrophoresis-convection Apparatus BY RAYMOND A. BROWN,^ JOHN B. SHUMAKER, JR., JOHN R. CANNAND JOHN G. KIRKWOOD A study has been made of the conditions affecting operation of the electrophoresis-convection apparatus for fractionation of proteins. Systems containing a single protein have been investigated for the dependence of the time of transport upon the electric field strength and the mobility, initial concentration, and fraction of protein transported. The experimental results show satisfactory agreement with the theoretical treatment of the rate of transport. The characteristic times of transport correspond to effective field strengths bearing ratios of between unity and one-fourth to the nominal applied field strength.
Introduction A systematic experimental investigation of the conditions determining the rate of transport of proteins by electrophoresis-convection is reported in the present article. The results demonstrate that the theory of transport may be used to obtain practical estimates of the time required to transport a specified fraction of a given protein from the top to bottom reservoir of the apparatus if a factor of safety between 2 and 4 is applied to the theoretical field strength. The electrophoresis-convection apparatus as described by Cann, Kirkwood, Brown and PlesciaIa consists of two reservoirs connected by a narrow, vertical, semi-permeable channel formed between two sheets of Visking Corporation sausage casing. The top of the upper reservoir is open so that the reservoirs and channel may be filled with a solution of the proteins to be fractionated. In operation the apparatus is filled and immersed in a suitable buffer solution between two flat platinum electrodes arranged to provide a homogeneous electric field across the channel upon the passage of a direct current between them. The field effects a horizontal transport of the protein components establishing horizontal density gradients in the channel, which, under the action of gravity, result in differential migration of the proteins into the lower reservoir. In the case of a single protein system the protein is simply concentrated in the bottom reservoir, External circulation of the buffer is maintained to prevent accumulation of electrolysis products. Kirkwood and co-workers2have shown that for a system containing a single mobile protein the time, t, required to transport a fraction 1 - y of mobile protein from the top reservoir is, for equal volumes, V , in top and bottom reservoirs t =
OI(-/)
where I ( y ) = (5/3)'/'
J1-T
[(I
- y)-'/5
- (1 -k y)-'/$\'/* dy (1)
and 8 =
2VD/hblp2E2,h = (4q1D)/cupgC')'/~
The integral I, equal to t / e , is presented as a function of 1 - y in Table I. In these expressions, (1) U. S. Public Health Service postdoctoral fellow of the National Institutes of Health. ( l a ) J. R. Cann. J. G Kukwood, R A Brown and 0 J. Plescia, THIS JOWRNU, 71, 1603 (1949). (2) J G.Kirkwood. J R Cann and R A Brown Rzochim Biophys L 5 . 301 (1960)
TABLE I TIME OF TRANSPORT AS A FUNCTION OF FRACTION OF MOBILF COMPONENr TRANSPORTED t/s
1-7
t /e
I--/
0.006 ,029 .074 .I45
0.10 .20 -30 .40 .50 .60
0.631 0.972 1.57 2.13 3.21 5.57
0.70 .80
.253 .407
.90
.95 .99 1 00
is the electrophoretic mobility; D,the diffusion constant; y, the fraction of protein remaining in the top reservoir; and cotthe initial concentration in grams per 100 ml. of the mobile component. p is the density and 7, the viscosity of the solvent, b is the channel width; I, the channel length; g, the acceleration of gravity; E , the electric field strength; and cup, the density increment produced by 1 g. of protein per 100 ml. of solution. This theory is derived under the conditions p
pEa/D
