Thin Film Dialysis

THIN F l l M DIALYSIS By KENT K. STEWART Nutrient Composition laboratory, Nutrition Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland

I. Introduction . . . . . . . . . . . . . . 11. Theory of Dialysis . . . . . . . . . . . . A. Mechanism of Dialysis . . . . . . . . . B. Parameters Affecting Dialysis Rates. . . . . C. Thin Film Dialysis . . . . . . . . . . 111. Experimental Methods . . . . . . . . . . A. Thin Film Dialyzers . . . . . . . . . . B. Dialysis Membranes . . . . . . . . . . C. Pitfalls and Artifacts in Thin Film Dialysis . . IV. Applications ofThin Film Dialysis . . . . . . A. Techniques in Purification and Separation . . B. Binding and Hydrogen Exchange Studies . . C. Measurement of Solute Size and Conformation V. Horizons in Thin Film Dialysis . . . . . . . References . . . . . . . . . . . . . . .

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169 172 175 181 182

INTRODUCTION There will be a net movement of solvent and solute molecules when a semipermeable membrane separates a solution from pure solvent. If the solution contains two or more solutes with different capacities to diffuse through the semipermeable membrane, a separation process can take place. This separation process is called dialysis, and it was first described by Thomas Graham (1861). He used this technique for the separation of small molecules (sucrose) from large ones (gum arabic), and, as a result of his studies, he classified solutes as either crystalloid (pass through parchment membranes and are crystallizable) or colloids (are retained by parchment membranes and are not crystallizable). These early observations provided part of the foundation for the study of the chemistry of biological materials and form a cornerstone of modem biophysical techniques and separation processes. For a long time, protein chemists viewed dialysis as a simple, useful tool for protein isolation and purification, and nothing more. In the I.

135

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KENT K. STEWART

1950s, Lyman C. Craig and his co-workers began a series of studies that has resulted in the demonstration that thin film dialysis can also be used as a specific analytical tool for the study of solute size and conformation in solution. The studies af Craig and others have brought the realization that analytical thin film dialysis can provide the detailed information required for understanding the solution chemistry of proteins and peptides. In this context it is pertinent to note that dialysis has been used concurrently with nuclear magnetic resonance, optical rotatory dispersion, and tritium exchange in studies of the structures of antibiotic peptides in solution (Burachik et al., 1970).

Thin film dialysis is discussed in this chapter with respect to theory, experimental methods, applications in preparative and analytical work, and finally, the future of the technique. The general topic of dialysis is not reviewed, except where it pertains to thin film dialysis. For reviews on dialysis, in general, the reader is referred to Ferry (1936a,b),Renkin (1955), Stauffer (1956), Sollner (1958), Cam (1961), Tuwiner (1962), Morris and Morris (1964), and McPhie (1971). Craig has written a number of reviews on thin film dialysis (1962,1964,1965, 1967, 1968; Craig and King, 1962; Craig et al., 1958, 1969).

11. THEORYOF DIALYSIS The present dialysis theory is not completely satisfactory. The qualitative aspects of the theory predict the dialysis behavior of solutes of similar chemical nature, but do not predict a priori the dialysis behavior of solutes of dissimilar chemical nature (Stewart and Craig, 1970). The current state of dialysis theory does offer some insights into the comparative dialysis behavior of solutes of the same or similar chemical classes, but it does not predict the quantitative aspects of dialysis with sufficient accuracy to be of much use for the protein chemist. Thus the quantitative aspects of dialysis are not covered in this chapter, and those readers who have particular interest in that aspect may find the following references to be of some help: Teorell (1935), Adair (1937), Mauro (1960), Kedem and Katchalsky (1958), Vink (1960, 1962a), Casassa and Eisenberg (1960), Ginzburg and Katchalsky (1963), Bresler and Wendt (1969), Yasuda et al. (1969), Michaels (1959), Friedman and McCally (1972), and Mikulecky (1972). A. Mechanism of Dialysis If a solution is dialyzed, its components are subjected to separation processes that are based upon differential diffusion rates through a

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137

semipermeable membrane. When a solute is dialyzable,’ it is capable of being purified b y dialysis. The diffuusate is that solution from the compartment which originally had the lower concentration of the solute of interest, and the retentate (Turner and Feinberg, 1959) is that solution from the compartment which originally had the higher solute concentration. The traditional dialysis cell consists of two compartments separated b y a membrane. At the start of a dialysis run, one compartment contains a solution of large and small molecules, and it is separated by the semipermeable membrane from the other compartment, which contains pure solvent. The membrane is generally chosen so that it is quite permeable to the small molecules and impermeable to the large molecules. Thus, with time, the concentration of the smaller solute will decrease in the retentate and increase in the diffusate while the larger solute remains in the retentate. If the solution in the diffusate compartment is replaced from time to time with pure solvent, then, eventually, the retentate compartment will contain only the larger solute. The solute’s net movement through the membrane is described by Fick’s law of diffusion (Fick, 1855), as is the case for all diffusion processes.

-dcldt = D,,A,(dc/dx)

(1)

D,, is the solute membrane diffusion coefficient, dcldx is the concentration gradient across the membrane, and A , is the cross-sectional area of the membrane. A , is defined as total area of the membrane times the fraction of the membrane available for solvent movement. Am = Atotal . (Asolvent/AtotaJ (2) When the dialysis cell is ideal, the concentration gradient across the membrane is equal to the difference in concentration between the two compartments. When the diffusate compartment contains pure solvent; then this difference is equal to the solute concentration in the retentate compartment, C,. The net movement of the solute is then described by Eq. ( 3 ) .

-dcldt = D,,A,C,

(3)

There is considerable disagreement as to whether or not a dialyzable solute will readily diffuse through a membrane (Williams, 1927). The author believes that the confusion over the termdialyzable is so great that individuals must define their usage of the term or, better yet, not use the term. A greater confusion exists about the word dialyzate. This term is used to refer either to the solution that contains the solute that did not diffuse through the membrane or to the solution that contains the solute that did diffuse through the membrane. As equal use has been made of both definitions, the author believes that the term dialyzate should not b e used.

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KENT K. STEWART

Rearranging and integrating from t

=

0 to t = t, w e get Eq. (4).

In( Cr,x=tlCr,x=O) = - D,A,t

(4)

The plot of the natural logarithm of the ratio of retentate concentration at time t to the retentate concentration at time zero versus the time is a straight line with a slope of - D,,A,. The membrane area can be estimated, and thus the solute membrane diffusion constant can be calculated. The differences in the solute membrane diffusion constants of different solutes are the basis for all dialytic separations and studies. B . Parameters Affecting Dialysis Rates The parameters that affect dialysis rates can be divided into those that are common to all diffusional processes and those that are unique to dialysis. 1 . Parameters Common to All Diffusion Processes Dialysis is a diffusional process and is subject to the same physical law as all diffusional processes. The physical process of diffusion is described by Fick's first law of diffusion [Eq. (l)],and the dependence of the diffusional coefficient (Dfree) on various parameters was described by Einstein (1908) [Eq. (5)]. I n this equation, R is the ideal gas constant; T , the temperature; N , Avogadro's number; 7,the viscosity of the solvent; and r, the radius of an ideal sphere related to the molecular volume of the diffusing solute.

Of particular importance to our inquiries on dialysis are the direct dependence of the Dwe on the temperature and the inverse dependence upon viscosity and molecular volume. Any conditions that affect the temperature, viscosity, or molecular volume affect the diffusion coefficient. Thus the study of the diffusion coefficient of peptides and proteins under different chemical conditions yields direct information on changes of molecular volume. This is an area of some interest to protein chemists. The special usefulness of dialysis is the differential effect of the relationship between the membrane d i h s i o n coefficient and the molecular volume of the solute of interest. The dialysis membrane amplifies the differences in the molecular volumes and thus provides a unique tool for separation.

2 . Parameters of Membrane Diffusion a. Pore Size. The mechanism of difhsion of solutes through membranes is not completely understood. However, the dialysis of most

THIN FILM DIALYSIS

139

solutes in aqueous solutions through the common membranes suggests that membranes act as mechanical sieves. Although the mechanical sieve approach to dialysis is an oversimplification and does not account for many observations including charge effects and solvent effects,2 it is a useful starting point for understanding those factors that do influence dialysis rates. The sieve model treats the membrane as a solid sheet of material perforated by a series of channels. In the simplest version, the membrane has a series of cylinders passing through it at right angles to the membrane surface. Ferry (1936a,b) postulated that to diffuse through a channel a molecule must pass through the opening without striking the edge. His equation for the effective area (Aeff)for diffusion of a spherical molecule through a perfect cylinder is given by Eq. (6). Aeff is determined by the difference between the radius of the pore ( a ) and the radius of the molecule ( r ) . A, is the total cross-sectional area of the pore. The equation is valid only for free diffusion of solutes through the pores and does not take into account the corrections needed for the solvent flow found in ultrafiltration. The reader is referred to treatises on ultrafiltration for further discussion of these effects. Computations with Ferry’s equations readily demonstrate that the potential for using dialysis rates to differentiate between solutes of similar size becomes greater and greater as the solute radii approach the dimensions of the pore radii (see Fig. 1). I n this figure the ratios of the effective area to the total area are plotted for different ratios of the solute radius to the pore radius as calculated from Ferry’s equation. It is apparent that the selectivity of the dialysis process is a function of the ratio of solute size to pore size. If the pore radii are much larger than the solute radii, altering the solute size has only a small effect on the dialysis rate. For example, increasing the solute radius 5-fold (from 1%to 5% of the pore radius) decreases the effective area of dialysis by only lo%, and thus there is very little selectivity. However, if the sol% In particular, it does not account for the process of diasolysis (Carr, 1961), in which the solubility of the solute in the membrane has a very strong influence on its diffusion rate. Brintzinger and Beier (1937) and Brintzinger and Gotze (1948) have described several experiments on diasolysis in which hydrophilic substances were separated from hydrophobic substances by diffusion of the hydrophobic substances through a hydrophobic membrane, such as rubber. Diasolysis may play some unknown role in diffusion through living membranes and has some obvious potentials for some sophisticated separation techniques. However, it is not the same as dialysis, and any further discussion of it is outside the scope of this chapter.

140

KENT K. STEWART

100(r/o)

FIG. 1. Calculated ratio of the effective area (Aeff)to the total area (A,) of a circular membrane pore as the radius of the solute (r)approaches the radius of the pore (a).

Ute size approaches the pore size, then small changes in pore size have large effects. For example, when the solute radius is increased from 90% to 95% of the pore radius, the dialysis rate is decreased by 75%. The Ferry equation is still used, but many workers view the effect as an exclusion phenomenon similar to that invoked in the theory of separation in gel permeation chromatography (Beck and Schultz, 1972). The magnitude of the postulated effect of geometric exclusion of Ferry's equation is not sufficient to explain the observed changes in the ratios of the membrane diffusion coefficients to the free diffusion coefficients of a solute when the ratio of the solute radius to the pore radius is altered. Lane (1950) suggested that a drag term similar to that in capillary flow be added to account for the observed behavior of membrane diffusion coefficients. The Renkin equation (Renkin, 1955) [Eq. (7)l incorporated the drag term. This equation gives a better fit to the observed diffusion phenomena for small solutes.

A/A, = (1 - rh)' [ l

-

2 . 1 0 4 d ~+ 2 . 0 9 ( r / ~-) ~0 . 9 5 ( ~ / ~ ) ~(7) 1

THIN FILM DIALYSIS

141

Beck and Schultz (1972) studied the diffusion of a number of solutes through membranes made from mica sheets bombarded with fission fragments from a 235U source and then etched with hydrofluoric acid. These membranes had essentially straight-through pores and were suitable for experimental testing of diffusion theory. Authors found that the Renkin equation was adequate at solute radius:pore radius ratios of 0.2 or less. However, it tended to underestimate the effect of decreasing the effective pore size of the membranes as the ratio became greater than 0.2 and the solute size approached the membrane pore size. Because the most useful region of thin film dialysis appears to be in that region where the ratio of the radii is greater than 0.2, the failure of the Renkin equation in this region leaves the student of thin film dialysis in a difficult position. The best theory available fails in the region where it is needed the most. Thus, at this time, there is no adequate theoretical basis for estimation of the effect on the dialysis rates of chemically similar molecules as their radii approach those of the pores of the membrane. However, the experimental evidence indicates that the diffusion process becomes more selective than predicted as the ratio increases. The selectivities demonstrated in Fig. 1 and predicted by the Renkin equation [Eq. (7)l are thus poorer than the observed selectivities (Beck and Schultz, 1972). At this juncture we are left with the problem of correlating the diffusion coefficient of the solute in solution with the membrane diffusion coefficient. The author believes that the best approach is shown in Eq. (8).

Dsm = (AedA,)

Dfree

(8)

Implicit in Eq. (8)is the assumption that the physical forces that underlie membrane diffusion are the same as those that underlie free diffusion and that the critical difference lies only in the effective area available for the solute to diffuse through the membrane. This assumption seems to be reasonable, and it remains to future workers to provide an adequate means of exactly determining AeR. Combining Eqs. (l),(2), (5), and (8) gives a general dialysis equation, Eq. (9):

which simplifies to Eq. (10):

dcldt = - a b d (dcldx)

(10)

where a is the constant term RIN 67r, b is the temperature and solvent

142

KENT K. STEWART

term T / q , and d is the term correlating the membrane effective area and the solute radii A,&-. Equation (9) predicts that the temperature-viscosity dependence on the rate of the dialysis of solutes that do not change their state of aggregation, hydration, charge, or conformation should be calculable. This prediction was verified in a dialysis study of small molecules with very highly acetylated cellulose membranes (Stewart and Craig, 1970). The predicted ratio of the diffusion coefficient at two temperatures is given by Eq. (11). When this ratio was calculated by use of measured viscosities of the solvent (0.1M potassium chloride) at 20°C and 40°C, the

DTJDT,= ( T l / T d ~ h i )

(11)

ratio was 1.64, which agreed with the experimentally determined values of 1.65 & 0.10. The data from these experiments are shown in Table I which is a combination of tables reported by Stewart and Craig (1970). Similar results have been found in other dialysis studies [see the corrected values for 0.2%tyrocidine A in Burachik et at. (1970)l.

This theoretical approach to dialysis was also confirmed by studies of Craig and Chen (1972) in which they demonstrated that the rate of dialysis of the solute inside the membrane was considerably slower than the dialysis rate of the solute out of the membrane. This obserTABLEI

Comparison of Dialysis Rates of Different Solutes at 20°C and 40°C" Mean half-escape timeb (minutes x lo-') Membrane

A

B

Solute

~H,O l4CH30H CH3'4CO0H ~H,O CH3l4COOH [I4C]Urea CH3I4COO-K' [I4C]Glycine D -[14C]Glucose [I4C]EDTA

20°C

40°C

0.15 0.32 0.52 0.11 0.23 0.40 16.0 23.0 29.0 28.0

0.087 0.18 0.29 0.07 0.14

0.26 10.0 13.5 20.0

19.0

Ratio of half-escape times at 20°C and 40°C

*

1.75 0.22 1.83 f 0.19 1.80 f 0.10 1.51 f 0.16 1.67 ? 0.06 1.56 2 0.04 1.63 f 0.12 1.71 f 0.24 1.46 2 0.16 1.62 -t 0.52

a From Stewart and Craig (1970). Reprinted with permission from Anal. Chem. 42, 1257-1260. Copyright by the American Chemical Society. The half-escape times were determined from the slope ofthe first-orderescape plots.

THIN FILM DIALYSIS

143

vation was evidence that the rate-limiting step in the dialysis was the diffusion of the solute into the membrane. These findings are consistent with the hypothesis that the effective area is the rate-limiting factor in dialytic diffusion. b. Pore Shape. One of the assumptions of the theories that have been discussed up to this point is that the pores of the dialysis membrane were cylindrical, which obviously is not always true. Nuclear magnetic resonance studies of solutes of the sizes able to diffuse through membranes indicate that in aqueous solutions these solutes are tumbling at a rate of approximately lo7to 10” times per second (Carrington and McLachlan, 1967). A simple consideration of the geometry shows that the effective diffusional diameter of a solute is directly related to its longest cross-sectional axis, that is, to the spherical volume described by this axis. Thus, the critical dimension of the pore is its minimum width. Obviously, a circle would have the greatest ratio of effective area to total area for the diffusion of a given solute. Craig and Konigsberg (1961) reasoned that if the pores in cellophane membranes were in fact circular, then the porosity of the membrane could be altered mechanically by stretching either in one direction (Fig. 2) to yield ellipses with lowered minimum widths, or

LINEAR AND CIRCULAR STRETCH I NG

FIG.2. Hypothetical pore shapes.

144

KENT K. STEWART 100 90 80 70 60 50

40 30

20

10 9 8

7 6

2

4

6

8

9

10 12 14 16 18 20

Hours

FIG. 3. Escape curves of ribonuclease with stretched membranes. From Craig and Konigsberg (1961). Reprinted with permission from]. Phys. Chem. 65, 166-172. Copyright by the American Chemical Society.

in two directions to yield circles with larger diameters. Some results of early studies on the effect of stretching the membrane on the dialysis rate are shown in Fig. 3. With the unstretched membranes the ribonuclease had a 50% escape time of 6.6 hours; when the membrane was stretched linearly such that there was an 18%decrease in the circumference of the membrane, the half-escape time increased to 17.5 hours. When the membrane was stretched both linearly and circularly, the half-escape time decreased from 6.6 to 0.9 hours. The results of these experiments demonstrate the importance of pore shape and are corroborative evidence for the sieve model of dialysis. c. Membrane Thickness. The accepted dogma in dialysis theory is that the dialysis rates are inversely proportional to the membrane thickness (Carr, 1961). Thus, for the most efficient dialysis, the membrane thickness should be minimized. Most workers strive to make their membranes as thin as possible without introducing mechanical weakness or nonuniformity. There have been some reports that the membrane thickness may not be as important as previously believed (Herbst, 1954; Craig and Chen, 1972). This is certainly true with the “skin type” membranes similar to those manufactured by A m i ~ o n . ~ Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture, and does not imply its approval of the exclusion of other products that may also be suitable.

THIN FILM DIALYSIS

145

d . Membrane and Solute Charge. Fixed charges on a dialysis membrane drastically alter the permeability of charged solutes through the membrane. This most interesting aspect of dialysis has not been studied to any significant extent in thin film dialysis systems, and most workers strive to remove all charge from the membrane (Chen et al., 1972). Thus, dialysis with charged membranes will not be discussed further in this chapter. Future studies in this area could be quite rewarding. Sollner (1958) and Tuwiner (1962) have reviewed the subject of ion-exchange membranes. A considerable amount of work has been done on ion-exchange membranes used in desalination studies. Dialysis studies of charged and uncharged solutes have yielded some interesting results. The charged solutes dialyzed through the uncharged membranes at rates equal to or less than those of uncharged solutes of comparable size (Table I). It is not known whether this is a general phenomenon. The mechanism for the reduced rate of dialysis through uncharged membranes is not completely understood. The reduced rate could be due to differences in hydrated molecular volumes or to some other mechanism, or be an experimental artifact due to undetected membrane charges. Highly acetylated membranes are known for their capacity to reject salt selectively b y the poorly understood process of reverse osmosis (Reid and Breton, 1959a,b). e. Donnan Equilibrium. Donnan (1911) was the first to point out that, when a semipermeable membrane is placed between a solution containing nondiffusible charged solutes and a solution containing permeable charged smaller solutes, the small ions will unequally distribute themselves on the two sides at equiIibrium. These concentration differences may be significant, and pH differences may occur (Tanford, 1961). The Donnan effect could play an important role in dialysis, if steps are not taken to minimize it. Fortunately, the difference in the ionic concentrations caused by the Donnan effect can be minimized by the presence of excess electrolyte on both sides of the membrane. This measure is often used where the Donnan effect could be a problem. f. Osmosis. The very nature of the dialysis process, whereby a solution is separated from a pure solvent by a membrane, establishes a situation where osmosis must be considered. The concentration gradient that is the source of the chemical potential for the net movement of the dialyzing solutes is at the same time the source of a chemical potential for a net movement of the solvent. The osmotic effect can become quite severe at high concentrations of dialyzing solutes, and Carr (1961)reported osmotic dilutions of23-fold. In analytical studies, the osmotic dilution may have to be taken into account. Shieh et al.

146

KENT K. STEWART

(1975) noted that osmotic flow effects can alter the shape of dialysis escape curves of solutes. Generally, the osmotic effects can be kept to negligible levels by using sufficiently dilute solutions. In preparative dialysis, almost all osmotic effects can be ignored. g . Ultrafiltration. Ultrafiltration can play a part in the movement of solute and solvent when the hydrostatic pressures on the two sides of the membrane are unequal. Renkin (1955) has reviewed ultrafiltration. More recently, Hoch and his co-workers (Hoch and Turner, 1960; Hoch et al., 1961; Hoch and Miller, 1966; Pusch and Wolff, 1974) have discussed in some detail the effect of ultrafiltration on solute diffusion. Ultrafiltration can become quite severe with closed dialysis bags, especially when extensive osmosis has taken place and considerable pressure has developed within the bag. Up to 10% ultrafiltration has also been observed in countercurrent dialysis (Craig and Stewart, 1965; Craig and Chen, 1969). Most thin film dialyzers have been designed to minimize ultrafiltration. h. Stirring. In most dialysis cells the volume of the two liquid phases is sufficiently large that the diffusion of the solute to and from the membrane can be important in determining the apparent rate of the diffusion of the solute through the membrane. Almost all workers agree that stirring will greatly facilitate the dialysis procedure, although some workers feel that the role of agitation has been overemphasized (Ogston, 1960). Some rather elaborate methods have been used for agitation of the solutions (Cabib and Algranati, 1960; Englander and Crowe, 1965), including several dialyzer designs that “rock and roll” (Kunitz and Simms, 1928; Lauffer, 1942; Stewart et al., 1962). More conventional approaches were used by Craig and King (1955) and Colowick and Womack (1969), and these seem to be adequate. C . Thin Film Dialysis Thin film dialysis is a process in which the retentate and diffusate layers are quite thin and are in intimate contact with the dialysis membrane. This configuration minimizes the contribution of free solution diffusion and can yield very efficient dialysis. Some interesting and ingenious designs of thin film dialyzers have been published: see Wood (1923), Seegers (1943), Saroff and Dillard (1952), Craig et al. (1957), Cabib and Algranati (1960), Englander and Crowe (1965), Craig and Stewart (1965), Katz and Walls (1968), and Zeineh et al. (1972). Most of these thin film dialyzers were designed for very rapid salt removal and/or buffer exchange and are characterized by very small ratios of retentate volume to diffusate volume (i.e., one to a

147

THIN FILM DIALYSIS

hundred to one to several thousand). Such dialyzers are efficient but do not lend themselves to those studies where it is desirable to measure solute concentration in both the retentate and diffusate or to collect the diffusate for further characterization. The volume of the difh a t e is simply too great to work with, except in quite specialized situations. Some thin film dialyzers that have higher ratios of retentate to diffusate volumes ( 1 : 1 to 1: 10) still are very efficient dialyzers, and can be readily used in those situations where it is desirable to measure or recover the solutes that have diffused through the dialysis membranes. The analytical dialyzers and the countercurrent dialyzers of Craig and his co-workers are the best known of the latter class of thin film dialyzers. The detailed construction and operation of these dialyzers is discussed in the section on experimental methods. In practice, the diffusion of ideal solutes follows the expected first-order kinetic behavior (see Fig. 4) described earlier in this section. It is instructive to examine the theoretical escape curves of mixtures of ideal solutes as well as the escape curves of nonideal solutes. Three common systems are represented in Fig. 5; ideal mixtures (Fig.

0.6

0.4

L t 0

P

i

10

I

20

Minutes

L

30

I

40

FIG. 4. Typical first-order plots: Dialysis of [14C]urea in 0.10 M potassium chloride, pH 5.5, dialyzed against 0.10 M potassium chloride; CI and CII duplicate runs at 40°C, CIII and CIV duplicate runs at 20°C. From Stewart and Craig (1970). Reprinted with permission fromdnal. Chern. 42,1257-1260. Copyright by the American Chemical Society.

KENT K. STEWART

148 inside A

A’

E

inside

inside

E (n-mer of A )

A

membrane

membrane

outside

outside

8’

membrane

B’ (n-mer of A ) outside

(a) (b) (4 FIG.5. Diagram of diffusing solutes. Concentrations of the solutes inside the membrane are A and B and the concentrations outside the membrane are A ‘ and B’. The first-order diffusion rate constants of solutes A and B are a and /3, respectively. (a) Diffusion of two ideal solutes through a membrane. The dialysis of each is first order. (b) Diffusion of two forms of one solute. The dialysis rates of the two forms are different although first order. The overall escape pattern is complicated by the A to B interconversion. (c) Diffusion of an aggregating solute. Solute B (an aggregate of A) does not dialyze, but can be dissociated to A. From Stewart et 02. (1970). Reprinted with permission from Anal. Chem. 42, 1252-1257. Copyright by the American Chemical Society.

5a), interconverting dialyzing monomers (Fig. 5b), and aggregating systems (Fig. 5c). Escape curves for each of these systems have been 1970). Equation (12)describes the forward calculated (Stewart et d., diffusion of a solute inside the dialysis tube. Equation (13) describes the back diffusion. The inside concentration is Ci and the outside concentration is Co and k is the diffusion coefficient of the solute.

-dC,ldt = kCi -dC,ldt = kCo The combination of these equations allows the calculation of the solute concentration inside and outside the membrane at any time during the dialysis. Experimentally in thin film dialysis, the back-diffusion problem has been minimized by using 10-fold larger volume outside than inside and b y periodically replacing the outside solution with fresh solvent. This treatment results in a close approximation to a linear plot of the logarithm of the percent of the solute remaining vs time (Fig. 6). Mathematically, the back-diffusion problem can be handled either by Vink’s method (Vink, 1962b) or by a series of calculations (Stewart et al., 1970) using a A t such that Eqs. (12) and (13)may be treated independently; recomputing C , and Co after each calculation. T h e latter method was used in the calculations for Fig. 6. The calculated escape curves for several ideal solutes and several mixtures of ideal solutes are shown in Fig. 6. Each individual solute

149

THIN FILM DIALYSIS

C

Time

FIG.6. (A) Calculated escape curves of three ideal solutes. Curve I is the escape curve of a solute with an a equal to 0.10; in curve 11, a equals 0.05; in curve 111, a equals 0.01. (B) Calculated escape curve for an equimolar mixture of solutes I and 111. (C) Calculated escape curve for an equimolar mixture of solutes I, 11, and 111. From Stewart et al. (1970). Reprinted with permission from Anal Chem. 42, 1252-1257. Copyright by the American Chemical Society.

has a straight-line first-order escape curve, but mixtures of solutes give an escape curve with an upward curvature. The escape curves of a nonideal solute in which two dialyzable monomers are interconvertible (Fig. 5b) were examined in three different situations: k, and k 2 (the interconversion rate constants of the solutes) much larger than, approximately equal to, and finally, much less than a! and /3 (the diffusion rate constants of the solutes). If k, and k, are much greater than a! and p, a family of straight lines is generated (Fig. 7), whose slopes are a function of the equilibrium constant for the A to B conversion. The same result is also observed for most of the situations where k, and k 2 are approximately equal to a! and p, as is shown in Table 11. Finally, when k, and k 2 are much smaller than a! and 6, upward curvature, such as that found with mixtures, is observed, as shown in Fig. 8. Escape curves of systems undergoing aggregation show considerably different behavior. The system in which a dialyzable monomer is in equilibirum with a nondialyzable aggregate (Fig. 5c) has a characterisitic downward curvature in its escape curve, as shown in Fig. 9. Examination of Fig. 9 and a number of other escape curves of aggregating systems has shown that the downward curvature is not always obvious in the escape curve of an aggregating system, but is observ-

150

KENT K. STEWART

50 30 0

20

-

5

5 0 E

:

10-

c

C

QI

Y 0, a

75 -

3-

2-

20

0

40

80

60

Time (minutes) FIG.7. Calculated escape curves of interconvertible dialyzable monomers, A and B, in rapid equilibrium. The dialysis rate of A is two-thirds that of B. Each line is labeled with the equilibrium constant of the A to B conversion. From Stewart et al. (1970). Reprinted with permission from Anal. Chem. 42, 1252-1257. Copyright by the American Chemical Society. TABLEI1 Dialysis of Interconuertible Monomers Where a and p Are of the Same Order of Magnitude as k, and k,"

&Q

0.1 1.o 5.0 5 .O 5.0 0.2 ~~~~~~

ki

k,

a

P

Apparent straight line?

0.02 0.055 0.55 0.055 0.0055 0.0110

0.20 0.055 0.11 0.011 0.0011 0.0550

0.02 0.0110 0.036 0.036 0.036 0.0110

0.20 0.0550 0.055 0.055 0.055 0.055

Yes Yes Yesb Yesh Yesb No (curve up)

~~~~~~

" From Stewartetal. (1970). Reprinted with permission from Anal. Chem. 42,12522257. Copyright by the American Chemical Society. The slope of the escape curve was the same in each of the plots.

151

THIN FILM DIALYSIS

I1 0

10

20

I

30

40

50

60

I

70

Time (minutes)

FIG.8. Calculated escape curves of interconvertible dialyzable monomers when A and B are rapidly dialyzing and slowly undergoing interconversion. Solute B dialyzes ten times as fast as solute A. In curve (A) the equilibrium constant is 0.1, and in curve (B) the equilibrium constant is 10.0. Froin Stewartet al. (1970). Reprinted with permission from Anal. Chem. 42, 1252-1257. Copyright by the American Chemical Society.

able when the appropriate combination of total concentration, equilibrium constant, and concentration range is present (Craig et uZ., 1965; Ruttenberg et al., 1966). Aggregating systems are the only systems known to the author which have escape curves that are concave downward. Two things should be pointed out with respect to the escape curve of the aggregating system. First, the curve should be examined over several half-lives to detect curvature (see Fig. 9). If the dialysis rate is observed for only a short period of time, the curvature will not be noted. Second, it should be noted that the apparent escape rate of the aggregating solute is always slower than the escape rate of the nonaggregating monomer. Thus, the shape of the escape curve in analytical thin film dialysis gives considerable information as to the state of the diffusing solutes.

152

KENT K. STEWART

Time (minutes) 2 Monomer; Monomer-Dimer ( x l 0 -

FIG. 9. Calculated escape curves of an ideal monomer and a concentrationdependent aggregating system whose monomer has the same dialysis rate constant as the ideal monomer. The dimer does not dialyze. Initial concentration was 0.1 M; the equilibrium constant is 5 x lo6;k, is 0.055 min-' and the monomer-dimer equilibrium is assumed to be instantaneous. Under these conditions, there is 99.9%dimer at the beginning of the dialysis and 99.9%monomer inside the bag when 99.9%of the solute has dialyzed out. The time scale for the aggregating system escape curve has been multiplied by lo-* to permit comparison of the escape curves. From Stewart et al. (1970). Reprinted with permission from Anal. Chem. 42,1252-1257. Copyright by the American Chemical Society.

If the escape curve is concave upward, then the solute behaves as though it were heterogeneous. The heterogeneity may be due either to mixtures of different solutes (i.e., an impure sample) or to the fact that the solute is undergoing a slow conversion between two or more dialyzable forms. The slow monomer-monomer interconversion can be easily distinguished from heterogeneous solutes. All that is required is to collect diffusate samples at different times, hold the samples to allow any equilibrium to take place, and then redialyze the

THIN FILM DIALYSIS

153

samples to determine the new escape curves. If the system is heterogeneous, then the fast-dialyzing sample will still be fast and the slow-dialyzing sample will still be slow. But, if a slow conversion is occurring, then the rapidly dialyzing samples should have escape curves that are similar, if not identical, to those of the slowly dialyzing samples. If the escape curve is linear, the sample is behaving as though it were homogeneous with respect to size. A linear escape curve cannot be used to determine whether or not a solute is monodisperse and ideal, aggregated over the concentration studied, or participating in monomer-monomer equilibrium where both monomers are dialyzable. It is possible, however, under some conditions, to distinguish between these three cases. If the system under examination is an aggregating system, dilution of the sample should decrease the amount of aggregate present. Thus, if solute escape curves are determined for starting concentrations over a 10- to 100-fold range, one should be able to find a range of concentration where the escape curve shows the classical downward slope of an aggregating system. The linear shape of the escape curves of monomer-monomer systems has a practical result-namely that these systems will often be overlooked. However, if other data indicate a system of this type, the linearity of the escape curves can be quite useful. The equilibrium system of monomer-monomer interconversion, such as that shown in Fig. 6, can easily be shown to have a first-order escape curve for the solute described by Eq. (14). The diffusion rates of solutes A and B are a and p, respectively;

Ci is a total inside s o h e concentration, and K,, is the equilibrium constant of the A to B conversion. Since the escape curves are straight lines even when a and p are in the same range as k, and kp,all these systems can be treated by the approximation that A and B are in rapid equilibrium. That is, whenever a monomer-monomer system gives a linear escape curve, the system may be treated as an equilibrium system. Thus, if a and p can be determined, the equilibrium constant can be calculated; likewise, if the equilibrium constant can be determined, then a and p can be calculated in terms of each other. If neither a,p, nor the equilibrium constant can b e determined by other means, then the method is limited. Although a series of linear escape curves may be generated under certain conditions, it becomes difficult, if not impossible, to determine whether there is only one species for each con-

154

KENT K. STEWART

centration or if there are two states with fixed diffusion rates with different equilibrium constants for each condition, or if there are multistates, each with its own diffusion constants and equilibrium constant.

111. EXPERIMENTAL METHODS The experimental apparatus and membranes used in analytical thin film dialysis are easily prepared, and the procedures used are easily learned and performed. These attributes make the process attractive, especially since so much information can be gained about molecular conformation and aggregation from analytical thin film dialysis. The method is one of the least expensive techniques available for such studies, and almost every laboratory can afford the materials required. The countercurrent dialyzers are more complicated and expensive, but they offer an extremely efficient means of dialysis. In studies with tritiated water, one pass through a counter current dialyzer reduced the total concentration of tritium by a factor of lo6 in less than 15 minutes (Craig and Chen, 1969). In the following section these dialyzers, their membranes, and the techniques of thin film dialysis will be presented and discussed.

A. Thin Film Dialyners 1 . Analytical Dialysis Cell The dialysis cell devised by Craig has evolved over some time from a series of dialysis cells (Craig and King, 1955; Craiget al., 1957; Craig and Konigsberg, 1961)to two of the present models (see Figs. 10a and lob). These dialysis cells are quite simple; however, they are capable of being used to determine diffusion rates through the membrane in a relatively short time and with good reproducibility. The cell design provides maximum dialysis area for a small volume of solution under such conditions that both the retentate and the diffusate are stirred. The cell consists of a glass collar, dialysis tubing, inside tube, outside tube, stirrup, line, and stirring motor. The glass collar is a section of glass tubing about 5 cm long, which has been carefully fire-polished at both ends. On this glass collar fits a section of dialysis tubing that extends approximately 10 cm below the glass collar, where it is tied off with silk surgeon’s thread. The inside glass tube fits inside the glass collar rather loosely and fits snugly inside the dialysis tubing so that the retentate volume of about 0.5 ml completely covers the inside membrane surface of the dialysis tubing and yet does not go up inside the glass collar. The outside glass tube is of slightly larger

155

THIN FILM DIALYSIS

15 r p m Inside tube

inside lube

Gloss collor

910s COIID~

Outside tube

membmne

0

b

FIG. 10. Schematic drawings of two thin film dialysis cells. (a) From Craig et al. (1969). (b) From Stewart and Craig (1970). Reprinted with permission from Anal. Chem. 42,1257-1260. Copyright by the American Chemical Society.

diameter and has a lip at its top of such diameter that the glass collar can easily fit inside. Approximately 5 ml of diffusate solution should cover the entire outside of the dialysis tubing. The diameters of the inside tube, glass collar, and the outside tube depend on the choice of membranes for the dialysis experiment. The apparatus is held by clamping the glass collar and attaching the other end of the clamp to a ring stand. The outside tube rests on a stirrup that is connected to a timing motor, with an eccentric, by a piece of fishing line. The movement of the eccentric on the timing motor causes the outside tube to be raised and lowered about 1 cm at each revolution. This raising and lowering exerts a pistonlike effect on the whole assembly, which causes stirring on the outside and on the inside by virtue of the flexibility of the membrane. The apparatus can be maintained at constant temperature either b y lowering the entire assembly into a constanttemperature water bath or b y fitting the outside tube with a water jacket and pumping water from the water bath through the jacket. The retentate solution normally is placed on the inside of the dialysis bag and the diffusate solution is placed on the outside. Samples are

156

KENT K. STEWART

taken by stopping the stirring motor and then removing the outside tube, pouring out the diffusate solution and adding an equal amount of fresh solvent to the outside tube and then replacing the tube on the apparatus. Alternatively, the sampling device shown in Fig. lob may be used. In this particular modification, the outside tube has a Luer tip attached at the bottom, and this, with the sampling syringe, facilitates the removal of the diffusate and its replacement with fresh solvent. This particular design is especially useful when the apparatus is fitted with a water jacket for temperature control. After the apparatus has been assembled and tested for mechanical correctness, the membranes should be washed with a series of changes of solvent until a low and stable baseline is obtained for the retentate and diffusate solutions. Almost all dialysis membranes contain a significant level of heavy metals as well as a material that has an absorption maximum at about 280 nm. Preparation of the membranes is discussed in a later section. Analytical dialysis can be performed in the following standardized manner. The solute is dissolved in the solvent and placed inside the membrane, and then the inner tube is inserted. Solution is added to the outside tube. At fixed sampling-time intervals, the diffusate is removed, set aside, and replaced with fresh solvent. At the end of the dialysis run, both the retentate and the diffusate are collected and set aside for analyses and the membrane is thoroughly washed and the washes are saved. The retentate, diffusates, and wash solutions are analyzed and the recoveries are calculated. Dialysis runs with low total recoveries or with high concentrations of the solute of interest in the wash solutions should be viewed with suspicion.

2. Thin Film Countercurrent Dialyzers The design of the thin film countercurrent dialyzer was first published in 1965 (Craig and Stewart, 1965), and the design for a modified version was later published by Craig and Chen (1969). This latter design was the basis for a commercial dialyzer which is now manufactured and sold b y Spectrum Medical Industries, Incorporated, of Los Angeles. A schematic drawing of the Craig and Chen thin film countercurrent dialyzer is shown in Fig. 11. The dialyzer consists of an inside spacer tube, an outside tube, a glass collar, the membrane, a drive system, a stand, and the appropriate pumps and tubing. The solution being dialyzed is pumped from the solution reservoir into the capillary tube of the inside spacer tube, down through the

THIN FILM DJALYSlS

157

Motor

Inside spacer lube

FIG. 11. Schematic drawing of a thin film countercurrent dialysis column. From Craig and Chen (1969). Reprinted from Anal. Chem. 41, 590-596. Copyright by the American Chemical Society.

capillary, out the bottom, and then up the space between the dialysis membrane and the inside spacer tube in a very thin film and finally to the space between the collar and the inside tube at the point where the inside tube becomes constricted. The retentate is then picked up by the peristaltic pump tubing and transported to a fraction collector or collection vessel. The dialysis solvent is siphoned into the top of the outside tube, moves down the dialyzer between the outside tube and the dialysis membrane in a thin film, and then is sucked out the diffusate exit at the bottom of the outside tube through a peristaltic pump and to a fraction collector or collection vessel. The inside tube is clamped to prevent its movement, and the outside tube rotates to maintain the uniform thin film on both the diffusate and the retentate sides of the dialysis membrane.

158

KENT K. STEWART

The inside spacer tube is generally about 100 cm long and has an outside diameter of 17 mm for most of its length, but decreases to 8 mm the last 10 cm of the inside spacer tube. Other overall dimensions may be used, but the outside diameter of 17 mm is particularly suited for use with standard No. 18 and No. 20 Visking dialysis tubing. A Becton-Dickinson male Luer joint is sealed to a l-mm capillary tube that runs the length of the inside of the tube; the glass collar is about 8 cm long and has an inner diameter (i.d.) of 18 mm. The membrane is attached to this collar, covers the inside tube, and is tied off at the end of the inside tube. The rotating outside tube is 97 cm in length of which the top 5 cm has an i.d. of 24 mm and the remainder has an i.d. of 15.6 mm. In the original design, a Becton-Dickinson male Luer joint was sealed to the bottom of this tube and was placed in a No. 18 Becton-Dickinson blunt syringe needle. The hub of the needle rode on a Teflon (Dupont) tube into which the needle fitted rather closely. This arrangement acted as the rotating bearing for the dialyzer. There have been several different unpublished workable designs of this bearing. Distances between the inside spacer tube and the outside tube are critical. A clearance of 0.5 f 0.1 mm between the glass tubes is satisfactory. A variable-speed motor provides the drive for the rotation of the outside tube. A four-belt, two-pulley system provides a means for rotation of this outside tube in a balanced manner. The tensions on the belts are adjustable so that increased friction from the dialyzer causes the belts to slide rather than tear the membrane. The overall design provides minimal friction or pressure on the membrane and is schematically shown in Fig. 11. When the system is properly adjusted, single membranes can often be used continuously for months, and, thus, many runs can be made on a single calibrated membrane. When it is necessary to dialyze under controlled temperature conditions, the dialyzer can be placed in a water jacket (not shown in the drawings) with the bottom bearing of the dialyzer passing through a rubber stopper. The top of the water jacket is open. A variable-speed pump, such as the Manostat pump sold by E. Griener and Company of New York, is used for the water circulation. Extremely efficient dialyzers result when Visking dialysis casing No. 20 or seamless celluiose casing No. 18 is used with the 17 mm i.d. inside tube. A satisfactory method for assembling the casing over the inside tube follows. One hundred centimeters of dialysis tubing is wetted, and checked for leaks. The diameter of one end is enlarged by pushing it over a tapered glass tube (Craig, 1965). This end is then

THIN FILM DIALYSIS

159

carefully pushed over the wetted glass collar nearly to the top. The membrane is temporarily held in place b y wrapping a rubber band around the dialysis membrane. The inside spacer tube is covered with glycerol, and 5-10 ml of glycerol are placed inside the dialysis tubing attached to the glass collar. The glass collar and tubing are then carefully and gently pulled over the inside spacer tube at a smooth, even rate. While the tubing is drawn over the glass spacer tube, a constant stream of water from a wash bottle is directed at the dialysis tubing at the point where it passes over the glass tube. This water provides a lubricating film both inside and outside the dialysis membrane, presumably by osmotic flow, and allows an even stretching of the membrane. When the membrane does not smoothly slip over the inside tube, it will have discontinuities that tend to balloon in the dialyzer and it will puncture. After the dialysis casing has been pulled over the inside spacer tube, it is kept wet and its position is adjusted so that the glass collar is 6-7 cm below its final position. The bottom of a dialysis tubing is then tied off with silk thread. The membrane is again wetted to ensure its easy movement on the glass tube, and the glass collar is moved up to its final position. The collar is secured by hooking two nylon threads from the metal collar on the glass collar to the metal collar at the top of the inner tube. The dialysis casing is thus under tension linearly as well as circularly. It is linearly stretched approximately 35%, and approximately 13% circularly. The assembly is then carefully inserted into the outer tube, taking extreme care to keep all SUTfaces wet. Its position is adjusted by the clamp at the top so that it hangs easily in the outer tube, which is filled with water. The outside tube must always be kept full of water so that the membrane is kept wet. The solvent and solution pumps are then set up and filled with water or buffer. The retentate and diffusate streams are then started at a flow of approximately 0.5 ml per minute, and the outside tube is slowly and carefully started rotating. A satisfactory membrane assembly will allow the passage of water through both the retentate and the diffusate system when the outside tube is rotating. At this point the membrane should be checked for leaks by injecting 1 or 2 ml of a solution of blue dextran into the retentate side. In a properly assembled dialyzer, blue dextran solution travels down the capillary tube and then up between the dialysis tubing and the inside spacer tube. The band should be evenly distributed around the spacer tube; it should travel up the dialyzer smoothly and uniformly with little tailing, and should be collected at the exit of the retentate stream in 3 or 4 ml of solution. Since blue dextran is a very high molecular-

160

K E N T K. S T E W A R T

weight polymer, none of it should diffuse through the membrane into the diffusate stream. The appearance of any blue dextran in the diffusate stream indicates leaks in the dialysis membrane. If this occurs, the membrane must be discarded and a new membrane put on the dialyzer. Once the membrane has been found to be leak-free, it is ready for calibration. If the membrane is kept wet and proper care is exercised during the dialysis, it can be used for many runs. Membranes have been used continuously for weeks at a time.

B . Dialysis Membranes Dialysis membranes are generally thin layers of polymeric material that contain a considerable amount of bound solvent. Thin film dialysis membranes should be flexible, have a uniform mechanical strength, low ionic charge, and a polarity compatible with the solvent to be studied. It is apparently essential that the membrane contain a considerable amount of bound solvent, which can be interchanged with a free solvent. Although there are a number of polymeric materials that could theoretically meet these requirements, almost all the thin film dialysis membranes have been prepared from seamless regenerated cellulose dialysis tubing manufactured by the Union Carbide Corporation. This tubing is manufactured primarily for the sausage industry, not for the specific purpose of dialysis. The technical requirements of the sausage industry are for a thin membrane with a uniformly high wet strength, with very few pinholes and a very low carboxyl group content. These are precisely the requirements for thin film dialysis. The cellulose membranes meet the other requirements for dialysis in aqueous solutions. We will limit the discussion of the dialysis membranes to a discussion of the cellulose membranes. Two types of cellulose casing are available: (1) the so-called “dialysis” tubing in the sizes Iisted in Table 111; and (2) the regenerated seamless cellulose tubings in sizes similar to those given in Table 111. The dialysis tubing is selected specially for dialysis and tends to have slightly larger pore sizes than the other tubing. Each size of dialysis tubing has a slightly different pore size and thus a slightly different porosity. Although there is some lot-to-lot variation, the extent of this variation is quite small and the porosity of different portions of tubing within one roll has been found to be remarkably constant. The dialysis tubings are usually marketed as continuous rolls of 50, 100, 500, or 1000 feet. They are shipped in sealed plastic bags to prevent them from drying out. Once the bag has been opened and some of the tubing has been removed, the remainder should be replaced in the bag, a small amount of water

161

THIN FILM DIALYSIS

TABLE111 Seamless Regenerated Cellulose Dialysis Tubing Size identity

Flat width (inches)

Wall thickness (inches)

8 Dialysis 18 Dialysis 20 Dialysis 23 Dialysis 27 Dialysis 36 Dialysis li S. S. Dialysis 3$ S. S. Dialysis

0.39

0.0020

0.98

0.0008

1.31 1.73 2.90-3.14 4.65-5.10

0.0009 0.0008 0.0016 0.0035

-

-

added, and the bag closed tightly and stored in the refrigerator. In a number of cases the same roll of cellophane dialysis tubing has yielded amazingly consistent dialysis rates even when it had been stored in the refrigerator for one or two years. Generally, however, there is a small but consistent decrease in the porosity in the membranes stored in this manner. The dialysis tubing is manufactured from regenerated cellulose obtained from cotton and has added glycerol as a plasticizer. The membranes usually contain approximately 0.1%of a number of unidentified sulfur compounds, which apparently cause the strong spectral absorbance at 280 nm. Heavy-metal contamination is common. Most of the 280 nm absorbing material can be removed by extensive soaking in 0.001 N acetic acid, and most of the heavy-metal contamination can be removed by extensive soaking in dilute EDTA. However, the possibility of the presence of other contaminating materials should not be excluded. Some workers use extremely vigorous procedures to clean u p the membranes [see, for example, the procedure recommended by McPhie (1971)l. These vigorous clean-up procedures can alter the porosity of the membranes and should b e used with caution. The membranes are susceptible to attack b y microorganisms, and it is advisable that some bacteriostatic compound b e added to the solutions used for storage. The porosity of the membranes is irreversibly altered if the membranes are allowed to dry out, so it is imperative for reproducible dialysis. studies that the membranes be kept wet. The dialysis tubing as it is obtained from the manufacturer is generally suited only for the thin film dialysis of solutes of molecular weights between 6000 and 20,000. The thin film dialysis of solutes outside this range requires modified membranes. The porosities of

162

KENT K. STEWART

TABLEIV Membranes Suitable for Thin Film Dialysis of Solutes of Different Molecular Sizes” ~~

~~

Visking casing

~~

~

Molecular weight range

~

~~

18 Untreated 18 Stretched 18 Stretched linearly and acetylated 20 Untreated 20 Stretched linearly and circularly under pressure 20 ZnCITtreated

~

6,000- 12,000 2,000-6,000 18-2,000 12,000-20,000 20,000-45,000 45,000- 135,000

a From Craig (1968) as modified by Stewart and Craig (1970). Reprinted with permission.

the membranes may be modified by mechanical or chemical treatment. In Table IV are listed the different membranes most suitable for the analytical dialysis of peptides and proteins of different molecular weights (Craig, 1968).

1 . Mechanical Alteration of Pore Size A diagram of equipment used for mechanical stretching of dialysis membranes is shown in Fig. 12. The appropriate length of a wet dialysis casing is slipped over the glass collar of the analytical thin film dialysis tubing and secured with rubber bands. The appropriate yoke is attached to the glass collar, and a loop of cord extending from this yoke goes around the hook of a “C” clamp and back to the other end of the yoke. A simple version of this yoke is supplied by several turns of a nylon fishing cord. The length of the looping cord extending over Hydrostatic pressure

Glass coiior

FIG. 12. Apparatus for stretching the membrane. From Craig and Konigsberg (1961). Reprinted with permission from J . Phys. Chem. 65,166-172. Copyrightby the

American Chemical Society.

THIN FILM DIALYSIS

163

the glass collars and around the “C” clamps is adjusted so that the glass collars and the dialysis tubing are taut when the “C” clamps are open. One glass collar is plugged with a solid rubber stopper, and a one-hole rubber stopper with a short length of tubing is placed in the other collar. A piece of flexible tubing is attached to the glass tube at one end and to a compressed air or nitrogen tank at the other end. To increase the porosity of the membrane, it is necessary to apply air pressure at the same time that the “C” clamps are tightened. Under these conditions it is possible to stretch the casing lengthwise and circularly in a measured way. The dialysis tubings may be stretched as much as 50%before a break occurs. The amount that the membrane can be stretched will vary from roll to roll. However, within a single roll of dialysis tubing, the strength of the tubing is generally consistent. As was mentioned previously, stretching can markedly increase porosity of the membrane. In one case the stretching of No. 20 tubing increased the porosity of the membrane so that the half-escape time of ribonuclease dropped from 6.6 hours to 0.9 hour for the membrane stretched linearly and circularly. If the experimenter desires to decrease the porosity of the membrane, hydrostatic pressure is not used and the membrane is stretched only linearly. In one experiment with ribonuclease, stretching the membrane changed the half-escape time from 6.6 hours to 17.5 hours with the linearly stretched membrane. Both stretching procedures can be done in a reproducible manner and are useful methods for altering porosity of the membranes. A difficulty of this procedure is that the final diameter of the membrane will often vary from run to run. This requires that matched individual glass dialysis inside tubes be used to maintain the desired volume-to-surface ratio in the thin film dialyzer. It is often necessary to individually select the outside tube in these situations. This requirement for individual selection of inside and outside tubes can be inconvenient, although it need not prevent a conscientious worker from using the technique.

2 . Chemical Treatment Chemical treatment of the membrane can be used either to increase or decrease porosity of the cellophane membrane. The appropriate chemical modification can also be used to add or remove charges from the cellophane membrane. Generally, chemical modification of the dialysis membranes has a greater flexibility, allowing one to change the extent of porosity of the membranes while still controlling the diameter of the final dialysis membrane. The simple clamping system described in Fig. 13 fixes the membrane length and prevents

164

KENT K. STEWART CI

inside tube glass collar CI

membrane

FIG. 13. Schematic drawing of a holder for the chemical treatment of the membranes. From Stewart and Craig (1970). Reprinted with permission fromdnal. Chem. 42,1257-1260. Copyright by the American Chemical Society.

unwanted stretching or shrinking during chemical modification. This type of apparatus can be used both for analytical thin film dialysis systems and for countercurrent dialyzers. Treatment of the membranes with zinc chloride or with the enzyme cellulase increases their porosities. Craig and Konigsberg (1961) reported several attempts to increase the porosity of thdse membranes by a crude preparation of cellulase from Aspergillus oryzae. They treated No. 20 Visking tubing with different amounts of cellulase for different periods of time. When they plotted the half-escape time of ovalbumin vs the cellulase units, they obtained a linear decrease in the plot. Although this method seemed to be promising as a means for increasing the porosity of the membranes, it was found that membranes thus treated were extremely fragile and extraordinarily difficult to work with. To the author’s knowledge, there have been no further reports of any attempts to increase the porosity of the membrane using cellulase; the preferred method is the zinc chloride treatment. McBain and Stuewer (1936) found that treatment of cellophane membranes with zinc chloride greatly increased the porosity of these membranes to solutes. This method has also been used to treat the cellophane membranes used in thin film dialysis. Craig and Konigsberg (1961) reported that when No. 20 dialysis tubing was treated for

THIN FILM DIALYSIS

165

15 minutes in a 64% zinc chloride solution, the membrane that resulted readily passed the dimer of human serum albumin, MW 134,000, while ribonuclease, bovine serum albumin monomer, and human serum albumin dimer had half-escape times of 15 minutes, 1 hour, and 3 hours, respectively. When the same membrane was treated for only 10 minutes, the escape time for the serum albumin dimer increased to 60 hours. Zinc chloride treatment of No. 23 dialysis tubing changed its porosity extensively. Before treatment, the dialysis of subtilin, MW 3100, gave a half-escape time of several hours; after treatment, this same membrane passed p-lactoglobulin, MW 35,000, with a half-escape time of 3 hours. However, in contrast to the zinc chloride-treated No. 20 membrane, the zinc chloride-treated No. 23 membrane still allowed only a very slow diffusion of the human serum albumin monomer, and the half-escape time was well beyond 60 hours. Zinc chloride-treated membranes have also been found to pass transfer RNA in sodium chloride solution (Goldstein and Craig, 1960). The zinc chloride-treated membranes have about the same physical characteristics, aside from the porosity, as untreated membranes. However, the membranes are very fragile during the treatment. Treatment of the cellophane membrane with acetic anhydride in pyridine decreases its porosity. Craig and Konigsberg (1961) found that acetylated unstretched membranes were stiff and difficult to work with. However, when they acetylated membranes that had been previously stretched longitudinally, they obtained good, usable membranes. When acetylation in 27% acetic anhydride in dry pyridine was carried out overnight at room temperature, the membranes would pass tryptophan and some small polypeptides with half-lives of 12-90 minutes, acetylation of the stretched membrane at 65"for 3 hours produced membranes that gave half-lives of 48 minutes to 23 hours. The data are shown in Table V. Craig and Ansevin (1963) extended the original work and studied the dialysis behavior of amino acids with stretched membranes that had been acetylated for 7 hours at 65°C. They studied size 18 and 23 membranes. With the acetylated No. 18 membrane, the half-escape times of amino acids in aqueous solution at 40" were 2.5 to 9 hours; and with the acetylated No. 23 membrane, the escape times were about 0.5 hour. There was a great selectivity in the diffusion of each of the amino acids and the amino acids could be distinguished by their half-escape times. Stewart and Craig (1970) reported the results of dialysis studies with highly acetylated cellophane membranes, where they treated the No. 23 stretched membrane with 25% acetic anhydride in dry pyridine at temperatures of 80"-90°C for periods of 1-4 hours. These membranes had very

166

KENT K. STEWART

TABLEV Escape Rates of a Series of Peptides in 0.01 N Acetic Acid through Stretched Acetylated Cellophane Membranesu Hal f-escape time Solute

(NH=ASO,

Tryptophan GlyTyr HisHis GlyGl yGly ValTrpValHis AlaAlaTrpGlyLy s ValPheValHisProPhe

AsnArgValPheValHisProPhe

Hypertension I (adecapeptide) a

25°C

MW

Acetylation

134 204 238 292 189 516 508 744 1016 1249

12 minutes 20 minutes 36 minutes 45 minutes 84 minutes 4 hours

-

65°C Acetylation 36 minutes 48 minutes 1.9 hours 4.1 hours 2 hours 9.9 hours 14 hours 23 hours -

-

From Craig and Konigsberg (1961). Reprinted with permission from]. Phys. Chem.

65, 166-172. Copyright by the American Chemical Society.

restricted diffusion. For example, the half-escape time of glycine was 2300 minutes, urea was 40 minutes, and tritiated water was 15 minutes. With these membranes the diffusion of water, methanol, acetic acid, and potassium acetate could all be distinguished (see Table I). Chen et al. (1972)reported that the treatment of dialysis membranes with water-soluble carbodiimides and glycine amide removed the residual carboxyl groups. This treatment caused significant changes in porosities of the membranes. Positively charged solutes had a decreased diffusion rate. In general, the effect of the removal of the residual charges on the membrane was to eliminate the apparent anomalies in diffusion rates that were observed when charged solutes were dialyzed in solutions of low ionic strength through untreated membranes. The treated membranes, in contrast to the untreated membranes, do not bind the charged solutes to any extent. The glycine amide-treated membranes also seem to have greater mechanical strength than the untreated membranes. Use of these membranes would probably prevent the calcium binding observed by Reed (1973) and remove the problems encountered in dialysis of low concentrations of pyrophosphate (Watson et aZ., 1962). The coupling of the glycine amide to the membranes is straightforward. The membrane is attached to a glass collar and prepared for treatment as previously described. It is washed sequentially with

167

THIN FILM DIALYSIS

0.5% sodium dodecyl sulfate, water, 0.5% Na, EDTA, and water and then suspended in 60 ml of 50/50 v/v dimethylformamide and 0.1 M cacodylic acid-sodium hydroxide buffer at pH 4.75 containing 1mmol of glycine amide. Ten milliliters of a solution of the same 50/50 mixture of dimethylformamide and cacodylic buffer containing 1mmol of the water-soluble diimide is added slowly with stirring. The reaction is allowed to proceed overnight at 25" or for 2 hours at 65". The membrane is washed for 30 minutes in 0.1 M acetic acid, followed by repeated washes with water, then 0.01 M NaCl until no further ultraviolet-absorbing material was removed in the washes.

C . Pitfalls and Artifacts in Thin Film Dialysis Generally thin film dialysis is quite simple and there are few problems for the experimentalist. However, there are a few pitfalls. It is always wise to keep a balance sheet of all the solute involved in

70 -

5030 -

*t I1

0

20

40

60

Time (minutes)

80

FIG. 14. Theoretical curves illustrating the effect of sampling time intervals on calculated escape curves with: fixed sampling time interval not greater than 60% of a sampling ); time interval increased by a fixed amount of half-escape time (M each sampling, to above 60% of a half-escape time (M); sampling time interval From ). doubled at each sampling to well beyond 60% of a half-escape time (U Stewart et al. (1970). Reprinted with permission from Anal. Chem. 42, 1252-1257. Copyright by the American Chemical Society.

168

KENT K. STEWART

the dialysis experiment. Excessive recoveries suggest impurities in the membranes or the occurrence of unwanted chemical reactions. Low recoveries suggest the loss of the solute due to precipitation or to binding to the membrane. In either case the dialysis data should be viewed with great suspicion. Pinhole membrane leaks are a constant potential problem. Routine checks are wise. A number of different test materials can b e used to test for pinhole leaks. The author's favorite is blue dextran. A more subtle pitfall is the effect of the sampling times on the escape curve of an ideal solute. Upward curvature can be generated simply b y increasing the times between each sampling. Figure 14 shows a theoretical treatment of this problem, and Fig. 15 shows an experimental verification of the phenomenon. The upward curvature

I

60

I

120

1

180

I

240

Time (minutes)

I

300

FIG. 15. Effect of sampling time interval on the escape curves of ['Tlurea. Duplicate experimental cuxves with sampling time intervals of 30 minutes (m and W). Experimental curves with the sampling time interval doubled after each W). , The half-escape time is approximately 30 minutes. sampling (M From Stewart et al. (1970). Reprinted with permission from Anal. Chem. 42, 1252-1257. Copyright by the American Chemical Society.

THIN FILM DIALYSIS

169

is caused b y back diffusion of the solutes and apparently becomes significant when the sampling time interval approaches 60% of the half-escape time of the solute. Upward curvature can also be caused by osmotic flow effects (Shieh et al., 1975).

Iv. APPLICATIONS OF THINFILMDIALYSIS Thin film dialysis can be used in almost any experimental situation in which more classical dialysis techniques are normally used. There are also a number of situations for which thin film dialysis is uniquely suited.

A. Techniques in Purification and Separation The traditional uses of dialysis are solvent exchange and removal of low-molecular-weight contaminants. Thin film dialysis can be quite useful in these operations. Small samples can be used, and equilibrium conditions are reached much more rapidly. The availability of modified membranes, which can be calibrated, gives the experimenter a choice of more selective membranes over a large range. Konigsberg et al. (1966) used the selectivity available in thin film dialysis to prepare salt-free tryptic peptides from f,-bacteriophage coat protein for lyophilization. Teipel and Hill (1971) used thin film dialysis to change buffers rapidly in their study of fumarase and its subunits. A number of workers used this technique to prepare samples for chromatography (Dickman et al., 1962; Greene et nl., 1963). In Paladini’s studies on bovine growth hormone, thin film dialysis was used to remove the free amino acids from the hormone (Santomb et al., 1966). Several workers have used thin film dialysis to isolate the lowmolecular-weight components from solutions containing a large amount of high-molecular-weight material. A number of workers have used thin film dialysis for the isolation of the products of enzymic digestion, Guidotti et al. (1962) used thin film dialysis to isolate tryptic peptides in their studies on the structure of human hemoglobin. Liu and Elliott (1965) used the technique to isolate the small fragment produced in the activation of streptococcal proteinase. Nolan and Smith (1962) isolated the free sugars released by treatment of the glycopeptides from rabbit 6-globulin by this method. Several workers have used thin film dialysis as a part of the endgroup determinations of peptides and proteins. Deutsch et al. (1961) used it as part of their determination of the carboxy-terminal residue of papain-produced peptides of human serum globulin. Kasper et al. (1965) used a similar approach in studies on subtilisin BPN. Hill and

170

KENT K. STEWART

Schmidt (1962) in their studies on the complete enzymic hydrolysis of proteins, used thin film dialysis to monitor leucine aminopeptidase and prolidase digestions. Wainfan and Hess (1960) used it in their studies of the enzymically active products of trypsin autolysis. Frater et al. (1965) and Karlsson et al. (1966) have also used the method in end-group assays. A number of workers have used thin film dialysis as a means of detecting solute heterogeneity. Examples are the studies of Englund et al. (1968) on ficin and the studies by Konopka et al. (1969) on the heterogeneity of Fe(ATP),. Thin film dialysis has also been used to estimate molecular weights of active compounds. Marfey et al. (1961) used it to estimate the molecular weight of luciferin, and King and Norman (1962) to study the molecular weight of ragweed allergens. Schally and Guillemin (1964) employed thin film dialysis to estimate the molecular weight of lysine vasopressin and to determine whether it existed in aqueous solution as a monomer or a dimer. Read et d.(1970) used this method to estimate the molecular weight of a hypotensive material in the tannin fraction of a crude isolate of Eucalyptus. The uses of thin film dialysis discussed thus far have all dealt with the analytical thin film dialyzer for small volumes of a sample. When large volumes of samples need to be dialyzed during an isolation procedure, countercurrent dialysis can be useful. Countercurrent dialysis was used by Millin and his co-workers (1969) in the isolation of tea flavor components. Lockett and Retallack (1972) used countercurrent dialysis for the isolation of a substance from blood that affected several functions. The differential removal of low-molecular-weight solutes, reported in an early study (Craig and Stewart, 1965) with the countercurrent dialyzer, is shown in Table VI. The solutions were dialyzed at 30 ml per hour against distilled water, which flowed at about 70 ml per hour. An untreated membrane was used. It is apparent that this procedure can be used to selectively desalt large volumes of a sample. Selectivity in the salt removal can be attained by adjustment of the membrane porosity, the dialysis temperature, the absolute flow rates, and the ratio of the diffusate and retentate flow rates. An interesting use of this dialyzer was the preparation of desalted urine protein samples as shown in Table VII. As expected, the urine from the patient with multiple myeloma (type 1) had more nondiffusible material. Most likely this material is Bence-Jones protein, and the method is a simple way to obtain a considerable amount of desalted peptide material for further studies. It should be pointed out that ancillary studies demonstrated that any given part of the material was in contact with the membrane for only about 10 minutes,

171

THIN FILM DIALYSIS

TABLEVI Dialysis Data Obtained with a Thin Film Countercurrent Dialyzer at 3°C" Percent removal Solute

Membrane size 18

Membrane size 20

84.7

84.3

67.0 46.5

52.8

19.0

31.5

97.7 92.8 89.6

99.0 97.9

Tryptophan (2.5 x 10-3 M ) Sucrose (6%v/v) Bacitracin M) (1.92 X Subtilin (2.42 x 10-3M ) NaCl(1 M) (NH,), SO, 3% satd (NH,),SO, 50% satd

-

" From Craig and Stewart (1965). Reprinted with permission from Biochemistry 4, 2712-2719. Copyright by the American Chemical Society.

Further studies (Craig and Chen, 1969) with the thin film countercurrent dialyzer demonstrated the efficiency of this apparatus. When tritiated water was dialyzed, the tritium level of the retentate was reduced at least six orders of magnitude. Preliminary studies were reported that were later expanded into an analytical method for aminoacylation of tRNA (Chen e t al., 1971). In this study the retentate to diffusate flow rate ratio was 7.5, and the system was an extremely effiTABLEVII Dialysis of Urine with the Thin Film Countercurrent Dialyzer"

Conductance

Weight

Retention solids (g/100 ml starting material)

0 92.7 98.0

0 96.1 98.7

2.831 0.111 0.038

0 95.4 98.9

0 85.3 90.8

2.856 0.419 0.263

Percent removal Sample Normal urine No treatment 1 Pass 2 Passes Myeloma urine No treatment 1 Pass 2 Passes ~

~

~

~

From Craig and Stewart (1965). Reprinted with permission from Biochemistry 4,2712-2719. Copyright by the American Chemical Society.

172

KENT K. STEWART

TABLEVIII Clearance of Amino Acids by Thin Film Countercurrent Dialysis".b Amino acids

Amount applied in 0.5 ml

Method of analysis

Alanine Leucine Lysine Histidine Tryptophan Tryptophan Tryptophan

0.2 pCi 50 pmol 50 pmol 50 pmol 5 pmol 200 pmol 200 pmol

Radioactivity Ninhydrin Ninhydrin Ninhydrin UV absorption UV absorption UV absorption

Amount remaining (%)

0.000 0.012 0.016 0.022 0.000 0.293' 0 .oo1= d

" From Chen e t al. (1971). Reprinted with permission from Anal. Chem. 43, 10171020. Copyright by the American Chemical Society. Diffusate buffer I, Flow rates: retentate, 0.42 muminute; diffusate, 5.6 ml/minute. was 0.01 M cacodylic acid-KOH, pH 6.0. Performed at faster flow rates: retentate, 0.83 ml/minute; diffusate, 3.0 ml/minute. 5% Polyethylene glycol was added in the diffusate solution.

cient means of removing amino acids (see Table VIII). The pattern of the retentate effluent is shown in Fig. 16. Use of this system yielded a dose response curve for the tRNA assay identical with that of the classical precipitation method.

B . Binding and Hydrogen Exchange Studies Dialysis has been used extensively in studies of the binding of small solute molecules to larger molecules. Thin film dialysis is well suited for these studies. It has been used for equilibrium as well as kinetic dialysis studies, and recently it has been successfully used in tritium-exchange studies. A brief overview of each type of use is presented in the following section.

1 . Equilibrium Dialysis The most common method used to study the binding of small mole-

cules to large molecules is equilibrium dialysis. The method is simple and inexpensive, and can be applied to a large number of different systems (e.g., see Klotz et al. 1946; Bush and Alvin, 1973). The particular advantages of thin film dialysis for studies of hormone and protein binding were recognized during the early development of thin film dialysis. Ginsburg and Ireland (1964) modified Craig's thin film dialyzer for this particular use, and their modified apparatus has been used by many workers. The study of hormone binding to the

173

THIN FILM DIALYSIS Aminoacylatlon assay of 1-RNA

by Dmlys~s

200 y

r

IC

0

20

30

40

50

60

70

60

Y

90

100

110

120

i 130

140

Tube No. ( 0 . 5 8 m l / + ~ b e )

FIG. 16. Pattern of retentate effluent for solutions containing ['CI-alanine and different amounts of tRNA. From Chen et al. (1971). Reprinted with permission from Anal. Chem. 43, 1017-1020. Copyright by the American Chemical Society.

neurophysins has perhaps seen the most extensive use of thin film dialysis (see Breslow and Abrash, 1966; Rauch et al., 1969; Furth and Hope, 1970; Breslow and Walter, 1972; Watkins, 1972; Plilka and Sachs, 1974). The binding of the various antidiuretic hormones was studied by Czaczkes et al. (1964). Stouffer and Hsu (1966) studied ACTH binding to proteins, and other hormone-protein binding has been studied by Hollenberg and Hope (1967a,b). Thorn (1965) studied the binding of calcium to proteins, as did Oldham et al. (1974). Bender and his co-workers (1975) studied, on a small scale, the binding of tryptophan by both animal and human serum albumin. Chen and Craig (1971) used the countercurrent dialyzer to study the binding of N-acety~-D-glucosamineto lysozyme. Under their conditions

174

KENT K. STEWART

equilibrium was reached in 10 minutes, allowing many binding experiments to be run in a day.

2. Kinetic Dialysis It was pointed out that equilibrium dialysis conditions are not required in the measurement of the binding of small molecules by macromolecules (Colowick and Womack, 1969; Womack and Colowick, 1973). The measurement of the rate of dialysis is sufficient to acquire the necessary data. Two approaches can be taken in kinetic dialysis studies of binding. One is to remove only negligible amounts of the ligand during the rate measurement, as Colowick and Womack did in their experiments. Under these conditions the equilibrium between the ligand and the macromolecule is not disturbed, and a series of measurements at different concentrations is needed to obtain the necessary data. The alternative procedure is to remove a substantial amount of the solute during the measurement. Deshmukh and Nimni (1969) used Craig’s thin film dialysis technique to measure the binding of a series of compounds to collagen by measuring the rate of removal of isotopic label by dialysis. Beyer et a2. (1972,1973) used this approach in their thin film dialysis study of 2-p-toluidinyl naphthalene-6-sulfonate (TNS) and its binding to peptides and proteins. Silhavy et d.(1975) developed the theoretical implications of this approach to the study of the binding of ligands by macromolecules. They reported that the exit of the ligand has a half-life porportional to (1 + P/&), where P is the concentration of the binding sites and Kd is the dissociation constant of the ligand and protein when the protein concentration is much greater than the ligand concentration. It would appear that the potential of thin film dialysis techniques in this type of binding study is considerable.

3. Hydrogen Exchange Studies In 1965, Englander and Crowe reported the use of rapid microdiaIysis in hydrogen exchange studies. Using a modified form of thin film dialysis, they achieved very rapid dialysis and were able to reduce the level of tritium present as tritiated water by a factor of a million in a few minutes. The data obtained in this manner were in excellent agreement with the data obtained by gel filtration procedures. The thin film countercurrent dialyzer has been used in a number of hydrogen exchange studies. Printz and Craig and their co-workers studied a series of different polypeptide antibiotics and hormones (Laiken et al., 1969; Printz et aZ., 1971, 1972; Galardy et al., 1971). Cambiaso and his co-workers (1970) used a similar approach for their studies of growth hormones. The high selectivity of thin film dialy-

THIN FILM DIALYSIS

175

sis, the extremely high efficiency of dialysis in the thin film countercurrent dialyzer, and the ease of the experimental technique make this an attractive method for hydrogen exchange studies. Laiken et al. (1969) found that the tritiated water concentration could be reduced to about one-millionth the original concentration in a 90-cm countercurrent dialyzer with an acetylated membrane, while 50-60% of the gramicidin being studied was retained. Cambiaso et al. (1970), in their studies of growth hormone with untreated membranes, completely removed 5 x los cpm per milliliter in a minimum dialysis time o f 2 4 minutes. The diffusate flow rates were a hundred times as great as retentate flow rates in their studies (12 vs 0.12 mvminute). C . Measurement of Solute Size and Conformation

To the author, the use of thin film dialysis in the studies of solute size and conformation is the most exciting and interesting application of this technique. The apparatus is inexpensive, the technique is simple, and the amount of information that can be acquired is impressive. The combination of thin film dialysis with hydrogen exchange studies and spectroscopic studies provides the protein chemist with an excellent set of tools for the understanding of the structures of peptides and proteins in aqueous solution.

1 . Theory AS was discussed previously, the general dialysis Eq. (10) is usefully divided into several parts.

dcldt

= -a

b d (dc(dx)

(10)

where a is the constant term RIN 67r,b is the temperature and solvent term T / v , and d is the term correlating the membrane effective area and the solute radius A,&-. As was discussed earlier, the effective diffusional diameter of a solute is most likely directly related to its longest cross-sectional axis. With peptides it would seem, a priori, that the length of this axis would be sensitive to changes in conformation of the peptide and that analytical thin film dialysis could provide the analyst with a sensitive probe of conformational change. Aggregation is also likely to change this critical distance and thus the dialysis rate of the peptide or protein. While the escape rate yields information on the solute size, the shape of the escape curve yields information on the dynamics of the solute interactions. The shape of thin film dialysis escape curves depends upon the relationship of the kinetics of the conformation changes to the kinetics of diffusion (see Section II,C).

176

KENT K. STEWART

2 . Studies of Peptide and Protein Conformation In the initial stages of their studies on conformation, Craig and his co-workers established that the dialysis rate was a sensitive function of size and that small differences in size could readily be detected.

1301

X

I

-1 I

*Trv

c

.t

0.8

5

0.9

L

.*/"a'

L

n 1.0

t

200 -

E

s?

180-

*Try *Tyr

177

THIN FILM DIALYSIS

For example, see their studies on the dialysis of amino acids (Craig and Ansevin, 1963). Solute charge had little effect, but artifacts could be created by solute adsorption. Figure 17 gives a summary of their findings. In their studies on the diffusion of sugars, Craig and Pulley (1962) calculated that changes of 2% in the molecular diameters could be detected by thin film dialysis. Further demonstration of the effect of molecular size on dialysis rate is shown in Table IX, where the halfescape times are tabulated for tryptophan and a series of peptides and proteins when an untreated Visking No. 20 cellophane membrane was used (Craig et al., 1957). These and other studies confirmed that the dialysis rates of solutes were related to their size and thus set the stage for the more sophisticated studies on molecular conformation in Craig's laboratory. Data from one of the earliest studies are shown in Fig. 18 (Craig et d . , 1958), in which the half-escape time of ribonuclease is plotted vs the ionic strength. It is obvious that the ribonuclease had a more compact structure at the lower ionic strengths. This work was extended to other proteins (see Chen and Craig, 1971). Guidottf and Craig (1963) TABLEIX Half-Escape Times of Various Polypeptides and Proteins" Half-escape time Solute

MW

Tryptophan Bacitracin Subtilin B Chain from insulin GI ucagon Insulin Cytochrome c Ribonuclease Lysozyme Trypsin Trypsinogen Chymotrypsin Chymotrypsinogen Pituitary lactogenic hormone Gliadin Ovomucoid Pepsin Ovalhumin

204 1,422 3,300 3,600 4,000 5,733 12,000 13,600 14,000 20,000 20,000 24,500 25,000 26,000 27,000 28,000 35,000 45,000

0.1 N Acetic acid

0.01 N Acetic acid 4 minutes 15 minutes 54 minutes 42 minutes 50 minutes 60 minutes 2 hours 2-3 hours 4 hours 6-5 hours 5 hours 9 hours 13 hours 29 hours 35 hours 80 hours More slowly than pepsin

" From Craig e t al. (1957). Reprinted with permission from J. Am. Chem. S O C . 79, 3729-3737. Copyright by the American Chemical Society.

178

KENT K. STEWART

1

0.oooi I I

0.0000!

I

0.01

0.001 Ionic strength

1 O.!

I

1

10

FIG. 18. Effect of ionic strength on half-escape time of ribonuclease. The solvent was 0.01 N acetic acid with added NaCl(0) or MgSOdO). From Craig et al. (1958).

studied the effect of p H and buffer composition (Fig. 19)and of solute concentration (Fig. 20) on the escape rate of carbonmonoxyhemoglobin. These studies demonstrate the wealth of information that can be gained from thin film dialysis, and they have an important place in the long series of experiments designed to elucidate the structure of

8 I0 12 PH FIG. 19. Effect of pH on half-escape time of carbonmonoxyhemoglobin in 0.2 M phosphate buffer (M and )in 0.4 M acetate buffer (MFrom ).Guidotti and Craig (1963). 4

6

179

THIN FILM DIALYSIS

0.0I

I

0.1

I

1.0

I

10

Starting conc. in g / 1 0 0 m i FIG.20. Effect of carbonmonoxyhemoglobin concentration on its half-escape time. w,0.2 M PO,, pH 7.1, + 2M NaCl; M, 0.2 M PO,, pH 7.1; U , 0.2 M KAc, pH 4.71. From Guidotti and Craig (1963).

hemoglobin. Craig and his co-workers (1965) extended these observations to conformational studies on a series of peptides. The results of their studies on the dialysis of ACTH, the A chain of insulin, and glucagon are shown in Fig. 21. It is interesting to note that all the types of escape curves predicted for pure solutes (see theoretical section) are represented in this figure. Note also the reversible nature of the glucagon size change. As has been mentioned earlier, the effect of temperature can be predicted theoretically by the Einstein equation, and this has been verified for small solutes (Stewart and Craig, 1970) and, in retrospect, for large solutes (Craig, 1965). Some peptides do not have escape curves that follow this equation, for example, 1-24 P-ACTH (see Fig. 22) (Craig, 1971). These curves indicate that the structure of 1-24 P-ACTH is thermally labile and undergoes a transition somewhere between 30°C and 50°C. The examples given here have shown that changes in conformation due to temperature, pH, ionic strength, buffer components, and solute concentration can be detected by thin film dialysis. Awareness of the technique’s potential has led a number of workers to use it to study the conformational changes of a number of peptides. The technique has generally been most fruitful when combined with spectroscopic techniques, although other techniques, such as binding studies and

180

KENT K. STEWART

20-

Change back to 001 H A c

-

1

1

1

1

1

1

I

ultracentrifugation, have been used. The reader is referred to the individual articles for more detail. General papers not previously mentioned include Craig et al. (1968, 1969, 1971, 1973, 1975) and Craig (1967, 1971). In addition, conformation studies have been reported on ACTH and its analogs, the insulin A chain, and glucagon (Craig et al., 1963), on ribonuclease (Craig et al., 1963), on lysozyme

181

THIN FILM DIALYSIS

E L

"\

3-

21

'

2 9 170°1

I

3 I (50’)

1

33130")

I

3 5110')

Reciprocol of temperolure ( K ) x I O - ~

FIG. 22. The effect of temperature on half-escape times. 0, Half-escape time, gramicidin SA; 0 , half-escape time, L-Leu-L-Try; X , half-escape time 1-24 P-ACTH; 0, free diffusion calculated from Einstein equation. From Craig (1971).

(Chen and Craig, 1971), on angiotensin, oxytocin, and vasopressin (Craig e t al., 1964), and on angiotensin I1 (Franze de FerLndez et al., 1968; Ferreira et al., 1969). Ruttenberg et al. (1966)repirted on studies of the tryocidins, as did Ruttenberg and Mach (1966)and Stern e t al. (1969). Hilschmann and Craig (1965) studied the dissociation of Bence-Jones protein under various solvent conditions. A series of studies on various synthetic peptides have been reported b y Burachik e t al. (1970), b y Birdi and Schack (1973), and by Harris and Craig (1974). Growth hormone has been studied by Paladini's group (see Dellacha et al., 1968a,b; Cambiaso e t al., 1970). Parathyroid hormone was studied by Rasmussen and Craig (1962), and edeine by Roncari et al. (1966). There have been some thin film dialysis studies of nonpeptide material; these include Goldstein and Craig's study on RNAs (1960)'and studies on sugars (Craig and Pulley, 1962; John et al., 1973).

v. HORIZONSIN

THIN FILMDIALYSIS

Thin film dialysis has not been used to its full potential in the investigations of peptide and protein structure. Too often, spectral studies have been the sole basis for reports on conformation changes in proteins. Often little or nothing is known about the order of the size change or whether or not aggregation has occurred with a change in solvent conditions. Yet obviously, some indication of the order of the size change can have very important consequences in the interpretation of the spectral data in the conformation studies. Analytical thin film dialysis has a place in these studies. The low cost of the appa-

182

KENT K. STEWART

ratus coupled with its ease of application should encourage investigators of peptide and protein structure to use this technique. The theory of thin film dialysis needs further development. One problem is the failure of the Renkin equation where it is most needed. Another is the apparent disparity in the membrane diffusion rates of charged and uncharged solutes. The need for an absolute means of determining the membrane diffusion constant is obvious. A great deal of work is needed on the detailed theoretical description of thin film countercurrent dialysis. The successful solution of any of these problems should encourage more use of the technique. Further development of the apparatus and membranes is also needed. The development of automated analytical dialyzers should be straightforward. These automated dialyzers should have many uses. Likewise, control ofthe membrane preparation is needed. Uniformity of membrane dimensions and more rigid control of pore size would be helpful. The development of membranes with different chemical matrices should be of considerable use.

REFERENCES Adair, G. S. (1937). Trans. Faraday SOC. 33, 1106-1116. Beck, R. E., and Schultz, J. S. (1972). Biochim. Biophys. Acta 225,273-303. Bender, D. A., Boulton, A. P., and Coulson, W. F. (1975). Biochem. SOC. Trans. 3, 193- 194. Beyer, C. F., Craig, L. C., and Gibbons, W. A. (1972). Biochemistry 11,4920-4926. Beyer, C. F., Craig, L. C., and Gibbons, W. A. (1973). Nature (London),New Biol. 241, 78-80. Birdi, K. S., and Schack, P. (1973). Macromol. Chem. 166,319-323. Bresler, E. H., and Wendt, R. P. (1969). Science 163, 944-945. Breslow, E., and Abrash, L. (1966). Proc. Natl. Acad. Sci. U.S.A. 56,640-646. Breslow, E., and Walter, R. (1972). Mol. Phannacol. 8, 75-81. Brintzinger, H., and Beier, H. (1937). Kolloid-2. 79,324-331. Brintzinger, H., and Gotze, M. (1948). Chem. Ber. 81,293-297. Burachik, M., Craig, L. C., and Chang, J. (1970). Biochemistry 9,3293-3300. Bush, M. T., and Alvin, J. D. (1973). Ann. N.Y. Acad. Sci. 226,36-43. Cabib, E., and Algranati, I. D. (1960). Nature (London) 188,409410. Cambiaso, C. L., Retegui, L. A., Dellacha, J. M., Santomb, J. A., and Paladini, A. C. (1970). Biochim. Biophys. Acta 221,290-296. Carr, C. W. (1961). Phys. Methods Chem. Anal., 4, 1-43. Carrington, A., and McLachlan, A. R. (1967). “Introduction to Magnetic Resonance,” p. 187. Harper, New York. Casassa, E. F., and Eisenberg, H. (1960). J . Phys. Chem. 64,753-756. Chen, H. C., and Craig, L. C. (1971). Bioorg. Chem. 1,51-65. Chen, H. C., O’Neal, C. H., and Craig, L. C. (1971). Anal. Chem. 43,1017-1020. Chen, H. C., Craig, L. C., and Stoner, E. (1972). Biochemistry 11,3559-3564. Colowick, S. P., and Womack, F. C. (1969). J . Biol. Chem. 244,774-777. Craig, L. C. (1962). Arch. Biochem. Biophys., Suppl. 1, 112-118.

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Craig, L. C. (1964). Science 144, 1093-1099. Craig, L. C. (1965). Adu. Anal. Chem. Instrum. 4,35-74. Craig, L. C. (1967). In “Method in Enzymology” (C. H. W. Hirs, ed.), Vol. 11, pp. 870-905. Academic Press, New York. Craig, L. C. (1968). Methods Zmmunol. Immunochem. 2,119-133. Craig, L. C. (1971). In “Structure-Activity Relationships of Proteins and Polypeptide Hormones” (M. Margoulies and F. C. Greenwood, eds.), pp. 447-454. Excerpta Med. Found., Amsterdam. Craig, L. C., and Ansevin, A. -(1963). Biochemistry 2, 1268-1271. Craig, L. C., and Chen, H. C. (1969). Anal. Chem. 41,590-596. Craig, L. C., and Chen, H. C. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 702-705. Craig, L. C., and King, T. P. (1955). 1. Am. Chem. SOC. 77,6620-6624. Craig, L. C., and King, T. P. (1962). Methods Biochem. Anal. 10,175-199. Craig, L. C., and Konigsberg, W. (1961).J. Phys. Chem. 65, 166-172. Craig, L. C., and Pulley, A. 0. (1962). Biochemistry 1, 89-94. Craig, L. C., and Stewart, K. (1965). Biochemistry 4, 2712-2719. Craig, L. C., King, T. P., and Stracher, A. (1957). J . Am. Chem. SOC.79, 3729-3737. Craig, L. C., Konigsberg, W., Stracher, A,, and King, T. P. (1958). Symp. Protein Struct. [Proc.], 1957 pp. 104-115. Craig L. C., King, T. P., and Crestifield, A. M. (1963). Biopolymers 1, 231-238. Craig, L. C., Harfenist, E . J., and Paladini, A. C. (1964). Biochemistry 3, 764-769. Craig, L. C., Fisher, J. C., and King, T. P. (1965). Biochemistry 4,311-318. Craig, L. C., Chen, H. C., Printz, M., and Taylor, W. I. (1968). N.A.S.-N.R.C., Publ. 1573, 315-329. Craig, L. C., Chen, H. C., and Harfenist, E. J. (1969). Prog. Sep. Purif 2,219-238. Craig, L. C., Kac, H., Chen, H. C., and Printz, M. P. (1971).I n “Structure-Activity Relationships of Proteins and Polypeptide Hormones” (M. Margoulies and F. C. Greenwood, eds.), pp. 176-180. Excerpta Med. Found., Amsterdam. Craig, L. C., Chen, H. C., and Gibbons, W. A. (1973). Adu. Chem. Ser. 125,286-297. Craig, L. C., Cowbum, D., and Bleich, H. (1975). Annu. Reu. Biochem. 44,477-490. Czaczkes, J. W., Kleeman, C. R., and Koenig, M. (1964). J. Clin. Inuest. 43,1625-1640. Dellacha, J. M., Santomb, J. A., and Paladini, A. C. (1968a). Ann. N. Y. Acad. Sci. 148, 313-327. Dellacha, J. M., Enero, M. A., and Paladini, A. C. (1968b). Biochim. Biophys. Acta 168,95-105. Deshmukh, K., and Nimni, M. E. (1969). J . Biol. Chem. 244,1787-1795. Dentsch, H. F., Stiehm, E. R., and Morton, J. I. (1961). J. Biol. Chem. 236,2216-2222. Dickman, S. R., Holtzer, R. L., and Gazzinelli, G. (1962). Biochemistry 1,574-580. Donnan, F. G., (1911). Z. Elektrochem. 17,572-581. Einstein, A. (1908). Z. Elektrochem. 14, 235-239. Englander, S. W., and Crowe, D. (1965). Anal. Biochem. 12, 579-584. Englund, P. T., King, T. P., Craig, L. C., and Walti, A. (1968). Biochemistry 7, 163-175. Ferreira, A. T., Hampe, 0. G., and Paiva, A. C. M. (1969). Biochemistry 8,3483-3487. Ferry, J. D. (1936a). J . Gem Physiol. 20,95-104. Ferry, J. D. (1936b). Chem. Rev. 18,373-455. Fick, A. (1855). Ann. Phys. Chem. 94,59 (from Tanford, 1961). Frame d e Ferngndez, M. T., Delius, A. E., and Paladini, A. C. (1968). Biochim. Biophys. Acta 154,223-225. Frater, R., Light, A., and Smith, E. L. (1965). J . Biol. Chem. 240,253-257.

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