Cycling Electrosorption
Cycling electrosorption in analogy to the thermal cycling zone adsorption process of Pigford and coworkers (1969a, 1969b) is proposed as a separation technique for biological molecules. Electrosorption equilibrium for ribonuclease A and glucoamylase on carbon was measured. Because of the tendency of proteins to irreversibly adsorb in many instances, it is suggested that the process may be more suitable for small molecular weight solutes such as amino acids.
Cycling Electrosorption The purpose of this note is twofold. First, we wish to call attention to an extension of the concept of separation by thermal cycling zone adsorption as developed by Pigford and others (1969a, 196913). The extension is the idea of effecting adsorptive separations by cycling the electrical potential at an adsorbing surface to vary the equilibrium amount of an adsorbed solute. Secondly, the idea of cycling electrosorption is particularly attractive for the separation of biological molecules (e.g., proteins) in that they are usually quite sensitive to temperature changes, denaturing or deactivating readily. Further, many such molecules carry a charge distribution which is a strong function of solution p H thereby providing an additional variable to affect the equilibrium amount adsorbed a t a given electrical potential. The process could also be carried out a t low temperature to preserve biological activity. We report here our measurements of the electrosorption equilibrium of the enzymes, ribonuclease and glucoamylase, on carbon in order to assess the feasibility of separation by cycling electrosorption; i.e., can enzymes be reversibly adsorbed to different degrees a t different electrode potentials? Electrosorption experiments on organic molecules which provide data on the variation of the amount of solute adsorbed with the potential of the charged surface have been conducted by a number of workers (Gileadi, 1965, 1967; Heiland et al., 1966). Electrosorption studies of fibrinogen on platinum by Stoner (1970) and fibrinogen on germanium by Mattson and Smith (1973) indicate that the amount of protein adsorbed varies with electrical potential, but these experiments were carried out a t very low protein concentrations.
Experimental Section T o obtain data on electrosorption, a cell was constructed containing two compartments connected by a tube allowing ion movement but no mass flow between the two compartments. Details may be found in Lee (1974). In one compartment was placed a thin, porous carbon electrode and a saturated calomel reference electrode, while in the other compartment was a platinum counterelectrode. The electrodes were connected to a potentiostat (Elron Model CHP-1) to set the desired potential which was measured with a Corning Model 10 pH meter. A solution of known volume and enzyme concentration was placed in the cell in contact with the carbon electrode. The desired potential was set and the solution enzyme concentration monitored as a function of time by uv absorbance at 278 nm until a steady value was achieved for 2 hr, indicating equilibrium had been obtained. The potential was then changed and another reading was taken. Experiments were conducted for a variety of solution concentrations and pH’s. The amount adsorbed was calculated by difference from the initial and final enzyme concentrations.
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Figure 1. Adsorption curve for ribonuclease A on carbon a t 25OC.
Results Figure 1 shows the adsorption isotherm of bovine pancreatic ribonuclease A (Sigma Chemicals) a t pH 5 at a potential of -150 mV relative to a standard calomel electrode (SCE). The adsorption was,Lhowever, totally irreversible in that changes in potential to as much as +300 mV SCE had no effect on the curve. The curve does resemble a Langmuir isotherm. Langmuirian adsorption of proteins on solid surfaces has been observed by Kemp and Rideal(l934) and Pollio and Kunin (1969). Experiments at other pH’s indicated irreversible adsorption, also. Our second series of experiments on the enzyme, glucoamylase (grade 11, Sigma Chemical), is summarized in Figure 2. Here, the adsorption was reversible and sensitive to etectrode potential in that switching of the potential from +300 mV SCE to -500 mV and back to +300 mV produced significant and reproducible changes in the amount of enzyme adsorbed except at the highest enzyme concentration observed. Experiments at p H 7.4 and 9.0, however, indicated irreversible adsorption. The curves in Figure 2 are particularly interesting in that they can possibly be considered ,as representing multilayer adsorption. Such multilayer adsorption behavior has been observed for enzymes a t the water-air interface (Yamashita and Bull, 1968; Khaiat and Miller, 1969). It seems reasonable to assume that irreversible adsorption of the proteins is due to denaturation (unfolding) of the molecule by an irreversible reaction of the protein with the surface. The existence of multilayer adsorption in the case of glucoamylase at pH 5.0 suggests that the binding of the protein to the surface is of a physical rather than a chemical nature. I t appears that because of the relative ease with which proteins may denature a t surfaces, the concept of electroInd. Eng. Cham., Fundam.. Vol. 14, No. 3, 1975
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slow diffusion rates of proteins within the porous carbon. On the other hand, the concept of electrocycling adsorption could perhaps be useful for separations of smaller molecular weight substances which do not denature (e.g., amino acids). Amino acid electrosorption is currently under investigation.
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Oileadi. E.. Ed., “Electrosorption,” Plenum Press, New York, N.Y., 1967. Gileadi. E., et el.. J. phys. Chern., 69, 3335 (1955). Heiland, W.. et ai., J. Phys. Chern., 70, 1207 (1966). Kemp, I., Rideal, E. K., Roc. Roy. SOC.,Ser. A,, 147, l(1934). Khaiit, A,, Miller, I. R., Biochem. Biophys. Acta, 183, 309 (1969). Lee, K. C., M.S. Thesis, University of Virginia, 1974. Mattson, J. S., Smith, C. A., Science, 181, 1055 (1973). Pigford.. R. L.. et al.. Ind. Eng. Chem., Fundam., 8, 144 (1969a). Pigford. R. L., et al., Ind. Eng. Chem., Fundam., 8, 848 (1969b). Pollii. F. X.. Kunin. R. “The Use of Macroreticular Ion Exchange Resins for the Fractionation and Purification of Enzymes and Related Proteins,” paper presented at Detroit AlChE Meeting, June 1969. Stoner, G. E., Srinivasan, S.,J. Phys. Chem.,74, 1088 (1970). Yamashtta, T., Bull, H. B., J. ColloidlnterfaceSci., 27, 19 (1968).
Figure 2. Adsorption curve for glucoamylase on carbon at 25T.
Department of Chemical Engineering University of Virginia Charlottesville, Virginia 22901
cycling adsorption would not be generally applicable to protein separations. Also, relatively long times (2-6 hr) were required to come t o equilibrium, no doubt due to the
Received for review November 18,1974 Accepted May 21,1975 This work was supported by NSF Grant GI-34772.
K. C. Lee D. J. Kirwan,
CORRESPONDENCE
Comments on the Theory of Active Transport through Human Cells
Sir: In the recent past, an interesting paper (Booij, 1969) outlined the difficulties in modelling active human cell transport, with special attention to the parametric pump model proposed by Wilhelm (1966). In the light of a recent theoretical result (Rice, 1973), two major points raised by Booij, those of length scale and high frequency, deserve reexamination. Booij poses the very important problem of scaling parapump models down from a laboratory apparatus of length 50-100 cm, to biomembrane thicknesses of 50-100 A. He also points out that a living cell is expected to have a very high frequency of oscillation (lo6 to 108 Hz), in comparison to the very low frequencies used in laboratory parapumps, typically for liquids of order Hz. At first glance, the combined problems of scale and frequency appear irreconcilable. However, in a recent paper (Rice, 1973) it was determined there may exist a simple relationship between fluid amplitude and frequency, pore size and molecular diffusion coefficient which maximizes the ultimate separation 200
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in a parametric pump. At the maximum ultimate separation, a simple dimensionless group was uncovered, namely AY,W/D
= 3
(1)
Assuming human cells operate near optimum, we use this simple expression to estimate the pore size, ro, corresponding to the.suggested frequency of lo7 Hz to see if a reasonable length scale evolves. Initially, let the fluid amplitude, A = ro and molecular diffusion is such that D * cm2/sec, so that 3 x 10-5 = 0.69 6.28 x 107
X
cm = 6 9 i
which is clearly in the range of (possible) human cell pore sizes, say 10-20 A. If we ask what is the optimum fluid amplitude when the pore size is say 20 A, again operating a t lo7 Hz, eq 1 shows A = 240 A, which is clearly in the range of the suggested biomembrane thicknesses of around 100 A.
