Sidney A. Katz, Charles Parfitt,' and Robert Purdy2 Rutgers University Camden, N e w Jersey 08102
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Equilibrium Dialysis A laboratory experiment
Equilibrium dialysis is a versatile technique frequently employed to study the interactions between compounds of high molecular weight and low molecular weight chemical entities. Wells ( 1 ) has employed this technique to study the binding of actinomycin by several DNA's. Cotton (2) has investigated the binding of nickel to protein with respect to allergic nickel dermatitis by equilibrium dialysis. The immunotoxicological problems associated with trivalent chromium have also been investigated by this technique ( 3 ) . I n the equilibrium dialysis method ( 4 , 5 ) ,the solution of high molecular weight compound is initially separated from the solution of low molecular weight compound by a semipermeable membrane. The characteristics of the membrane are such that it is permeable to the low molecular weight compound and impermeable to the high molecular weight compound. After equilibrium is established, the activity of the free low molecular weight compound is the same on each side of the membrane. When binding occurs, the low molecular weight compound is essentially removed from the system and does not contribute to the activity. 'Under these conditions, the total amount of the low molecular weight compound on each side of the membrane is no longer the same, and the difference is a measure of the extent to which the low molecular weight compound is bound by the high molecular weight compound. For a low molecular weight chemical species (M) interacting with a high molecular weight compound (P), the following equilibria can be considered P+M=PM P M + M = PMz
ki = (PM)/(P)(M) kz = (PM2)/(PM)(M)
The experimental parameter describing the extent of binding is r
=
Mb/Pt
where Mb is the number of moles of low molecular weight material bound and Pt is the total number of moles of high molecular weight material in the system. The quantity Pt is Bnown from the composition of the system and the quantity Mb is readily determined from the difference in the total amounts of low molecular weight compound on each side of the membrane a t equilibrium. The parameter r is related to the equilibria as
Present address: Whitehall Laboratories, Hammonton, N. J. Present address: Gateway Regional High School, Woodbury Heights, N. J.
The individual constants are interdependent and can be expressed as a single intrinsic constant, K, such that
The experiment described in this report deals with the binding of mercury(I1) by human serum albumin. Radiometric analysis was employed to determine the equilibrium amounts of mercury. The experiments were carried out in special cells designed by Icatz and Weisgerber (6). These cells were fabricated from 1-in. thick plastic (acrylate and methacrylate) sheet at a materials cost of less than $5 each. Similar cells are currently available from the Chemical Rubber Co. (15901 Cranwood Parkway, Cleveland, Ohio 44128) and Techni Lab Instruments, Inc. (Pequannock, N. J. 07440). The human serum albumin was purchased from Mann Research Laboratories. This material was reported to be 99% pure, and it was used as a 1.00 X lop5 M solution in 1.00 X lo-' M potassium nitrate buffered a t pH 4.0 with phosphate. The mercury(I1) solution was prepared from reagent grade Hg(N03)2.H20and high specific activity 203Hg. This solution, 1.00 X M with respect to mercury and containing 5 X 10-I microcuries 203Hg/ml, was made up in 1.00 X 10-I M potassium nitrate buffered a t pH 4.0 with phosphate. The equilibrium dialysis cells were fitted with sheets of Visking casing (A. H. Thomas Co.) and assembled. Five ml of the human serum albumin solution was introduced to one side of the membrane, and a measured quantity of the mercury(I1) solution and sufficient 1.00 X 10-I M potassium nitrate solution buffered a t pH 4.0 to give 5 ml was introduced to the other side. One ml samples were withdrawn from each side of the membrane after one week. The radioactivity of each sample was measured in a well counter, and the total mercury content of each sample was calculated in relation to the radioactivity of diluted aliquots of the original mercury(I1) solution. The results of our measurements are presented in the figure as the number of mercury (11) ions bound per molecule of human serum albumin against the free mercury(I1) ion concentration. Inspection of the figure indicates that one molecule of human serum albumin binds a maximum of 26 mercury(I1) ions. The double reciprocal plot ( l / r versus l/C) indicates that 27 mercury(I1) ions are bound/molecule of protein with an intrinsic constant of lo6. The nonlinearity of Volume 47, Number 7 0, October 7 970
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this plot suggests significant interactions between binding sites. This experiment is useful in demonstrating the technique of equilibrium dialysis and in introducing the complexity of protein-metal ion interactions. I n addition, the use of radiotracers allows direct measurement of the equilibrium amounts of the diffusable species without correcting for the "bag binding" factor.
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Literature Cited
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CONC. M(U) 1 0 0x lo6
150
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Results of measurements showing a maximum of 26 Hg ions bound to a molecule of human serum albumin.
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Journal of Chemical Education
(1) WELLS,R. D., Science, 165, 75 (1969). (2) COTTON,D. w. K., B ~ i tJ. . Dermatol., 7 6 , 99 (1964). (3) PIERCE,J. O., A N D STEMMER, K. L., Arch. Enuiron. Health., 1 2 , 190 (1966). (4) ALEXANDER, P., AND BLOCK,R. J., (eds.). "A Laboratory Manual of Analytical Methods of Protein Chemistry," Pergamon Press, N. Y., 1960, Vol. 11, p p . 141-151. (5) GLICK, D., (ed.), "Methods of Biochemical Analysis," Interscience (division of John Wiley & Son, Inc.) New York, 1956, Vol. 111. (6) KATZ,S. A., AND WEISGERBER, H. E., Ezperientia, 2 5 , 672 (1969).
