ARCHIVE8

AND

OF BIOCHEMISTRY

Equilibrium

173, 495-516 (1976)

BIOPHYSICS

Centrifugation

Beta-Lactoglobulin

of Proteins

A in Aqueous

SARA SZUCHET

Acetic,

AND

DAVID

in Acidic

Propionic,

Solutions

and Butyric

Acids

A. YPHANTIS’

Departments of Biology and Biophysical Sciences, State University of New York at Buffalo, Buffalo, New York 14214 and Biochemistry and Biophysics Section, Biological Sciences Group, and Institute of Materials Science, The University of Connecticut, StOFFS, Connecticut, 06268 and Departments of Biology and of Biophysical Sciences, State University of New York at Buffalo, New York 14214 Received

October 6, 1975

Short chain aliphatic acids are almost neutrally buoyant in aqueous solutions, and preferential interaction of macromolecules with these solvent components should not greatly affect apparent molecular weights determined by equilibrium ultracentrifugation. The feasibility of molecular weight estimations using native, neutral pH values of partial specific volume has been tested: equilibrium ultracentrifugation of /3-lactoglobulin A (fi-LgA) has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. These solutions are highly nonideal because of the extreme Donnan effect. Apparent molecular weights estimated at infinite dilution using the native neutral pH value of the partial specific volume, o,, differed by less than 5% from the monomer formula weight. The 10 M acids appear to be least effective as dissociating agents for /3-LgA, with a weak reversible monomer-dimer association suggested in 10 M acetic acid, with significant heterogeneity apparent in 10 M propionic acid, and with a lack of direct solubility in 10 M butyric acid. All the 0.1 M acids and all the 1 M acids were essentially equally effective as dissociating agents, with the exception of 1 M butyric acid which dissolved /3-LgA only slowly to give significantly heterogeneous solutions. From these results and from our previous experiments with aldolase (6), it appears feasible to use the native values of flP to obtain estimates of molecular weights of proteins in aqueous organic acids as dissociating agents.

A variety of reagents will dissociate proteins into subunits (1, 2). However, not all these reagents are equally suitable for the estimation of molecular weights and of interaction of subunits by equilibrium sedimentation methods. The most widely used reagent probably is guanidine HCl and, to a lesser extent, urea. Since these agents usually are only effective at high concentrations (6-8 M), complications may arise, most notably from preferential interaction of solvent components with the proteins, 1 To whom all correspondence should be addressed. Present address: David A. Yphantis, Box U-125, University of Connecticut, Storrs, Conn. 06268.

but also from other factors. For example, it has become common practice either to neglect or to assume an arbitrary correction factor [such as an assumed change in the partial specific volume, &,, of the protein ranging from 0.004-0.01 ml/g (3)] in order to account for preferential interaction in the presence of guanidine HCl. However, it has been shown recently by Lee and Timasheff (4) that such practices may be hazardous. Thus, while this correction appears to be adequate for certain proteins, it may lead to gross errors for others. Furthermore, after careful analysis of sedimentation equilibrium experiments of proteins in concentrated guanidine HCl solu495

Copyright 0 1976 Academic Press, Inc. All rights of reproduction in any form reserved.

496

SZUCHET

AND

tions, Munk and Cox (5) have drawn attention to the insensitivity of such systems to subunit heterogeneity. Thus, sedimentation equilibrium experiments in concentrated guanidine HCl or urea may not be particularly useful for answering questions of identity or nonidentity of protein subunits. We have proposed a different approach to the study of the quaternary structure of proteins-the use of aqueous solutions of acetic acid without any added electrolyte as dissociating agents (6). The rationale for this choice has been discussed at length; briefly, the selection was based on the expectation that for a mixed aqueous solvent with density close to that of H,O (component l), and therefore, with the buoyant mass of component 3, the added solvent component, close to zero (i.e., with M, (1 - 0,~) = 0), the effect of preferential interaction with solvent components would be minimized and the behavior of the system should approach that of a twocomponent system. For such systems the extrapolated value of the apparent molecular weight will reflect the value of M2, the molecular weight of the sedimenting component alone (7, 6). Moreover, in the case of aldolase in aqueous acetic acid, the errors introduced by the use of the partial specific volume of the native protein b,, for the calculation of molecular weights appear to be small. This was particularly true for the lower concentrations (5 1 M) of acetic acid but at higher concentrations (10 M) a somewhat larger error (-6%) was incurred by this assumption (6). Thus, in the case of aldolase in aqueous acetic acid, it was possible to sidestep the determination of 4 (8) and still obtain reasonably accurate estimates of molecular weights. Similar observations have also been made for myosin subunits (9). The term (1 - ti3p) is even closer to zero for other acids in the homologous series: propionic, butyric. Therefore, these acids may prove to be better solvents for molecular studies. Aqueous acetic acid has yet another feature, shared only partially by its shortchain homologs; namely, it does not appear to disrupt the secondary structure of proteins to the same extent as do guani-

YPHANTIS

dine HCl or urea (10). This can be surmised from the fact that proteins and peptides dissolved in aqueous acetic acid can partake in reversible associations (11, 12). Likewise, a biological active dimer has been recovered from bovine pancreatic ribonuclease lyophylized from 50% acetic acid (13). Thus, such dissociating systems may also yield interesting information on subunit interactions. Unfortunately, this did not prove to be the case for aldolase; the nonideality of the system was too complex to be accounted for in terms of a simple model (6). The main objective of this work was to establish a general procedure for molecular weight measurements of proteins under dissociating conditions which would not require determination of 4’ (i.e., the value of cp could be used instead) but which would still be of useful accuracy. Aqueous solutions of acetic acid appear to fulfill this requirement within 10% as a conservative estimate (6). Here we examine aqueous propionic and butyric acids as further solvents for that purpose. In addition, we were also interested in the interactions taking place in these highly nonideal systems, since these may have a bearing on subunit interactions in the native molecule. Furthermore, such mixed aqueous aliphatic acid solutions might be of use for the study of proteins that are only sparingly soluble in neutral solvents (e.g., membrane and structural proteins). The complex behavior of aldolase in acetic acid prompted us to look for simpler and better characterized protein systems. Two proteins were chosen for these studies. The first of these, p-lactoglobulin c/3LgA)2, is perhaps one of the best characterized proteins and its dissociation and association properties have been determined over a wide range of experimental conditions. Of particular significance to us was the fact that this protein is known to dissociate reversibly at acid pH apparently with little disruption of the native conformation of the individual subunits (14-16). Furthermore, the interaction of /3-LgA with 2 Abbreviations

used: p-LgA,

p-lactoglobulin

A.

CENTRIFUGATION

OF LACTOGLOBULIN

different organic solvents has also been investigated extensively (4, 17, 18). The amino acid composition of P-LgA is known (19, 20) and, more recently, the amino acid sequence has been determined (21). Similarly, ovalbumin is a well-characterized protein. We were particularly attracted by the fact that volume measurements have been made for ovalbumin under experimental conditions not too different from ours (22). This work has confirmed an observation made in our studies with aldolase; namely, that the accuracy obtained using the native partial specific volume, B,, for calculation of molecular weight decreases as the concentration of acid increases. This appears to be the case here for all three acids used. We have observed no differece in the accuracy of the estimated molecular weights using acetic acid or propionic acid at equivalent concentrations. It appears that even lo-~ solutions of either acid should yield reasonably accurate molecular weights. The solubility properties of some proteins differ in these acids, however. The following points have also emerged. (a) Each protein exhibits a characteristic behavior that appears to be determined primarily by the concentration of acid and to a lesser extent by the type of acid. (b) For the proteins used in these studies, the solubilities in the aqueous solutions of the acids investigated decrease as the length of the hydrocarbon chain increases. Here we present the results of our studies with /3-LgA. Our results with ovalbumin will be presented in a subsequent publication. MATERIALS

AND

METHODS

Materials. The fi-lactoglobulin A (/?-LgA) used was a six-times-recrystallized sample from genetically pure cows and was donated by Dr. S. Timasheff and by Dr. R. Townsend. This protein was further recrystallized by the method of Timasheff (private communication); the crystals were suspended in distilled H,O, and then just enough NaCl was added to dissolve the /3-LgA. This solution was clarified by centrifugation, then titrated to the isoelectric point

IN ACID

497

of pH 5.15, and finally dialyzed against distilled H,O in the cold. The solution was “seeded” with a few crystals to initiate crystallization. For each experiment the dried crystals were dissolved in the desired aqueous acid solvent and passed through a column (100 x 1 cm) of Sephadex G-25 equilibrated with the same solvent. The effluent protein solution was used directly (after adjusting the concentration) in the equilibrium experiments. All solutions contained 9.1% sucrose to provide a small stabilizing density gradient in the ultracentrifuge. A detailed description of the procedure followed has been given (6). Acetic, propionic, and butyric acids at concentrations of 0.1, 1, and 10 M were used. These acids were analytical reagents. The solubility of 6-LgA in acid decreased as the concentration of acid was increased from 0.1 to 10 M aqueous solutions. The solubility also decreased as the length of hydrocarbon chain of the acid increased; p-LgA dissolved readily in 0.1 M aqueous solutions of the three acids. The protein also dissolved rapidly in 1 M aqueous solutions of acetic and propionic acids; however, it took about 8 h to dissolve most of a sample of p-LgA in 1 M aqueous butyric acid, and even after that time there was a little precipitate which was eliminated by centrifugation. Crystals of P-LgA dissolved slowly but completely in 10 M aqueous acetic and in 10 M propionic acid; several attempts to dissolve p-LgA directly in IO M butyric acid proved completely unsuccessful. Dialysis tubing from Union Carbide was used and was pretreated by boiling in 5% NaHCO,. It was then washed with glass-distilled water and boiled in water to remove the NaHCO,. The tubing was stored in 0.01 M Na,EDTA (-pH 6) in the cold. Before use the tubing was thoroughly washed with glass-distilled water and soaked in the corresponding acid as required. Equipment and procedures. The Spinco Model E analytical ultracentrifuge used was equipped with electronic speed control, with schlieren, Rayleigh interference, and ultraviolet scanner optics, and with a temperature-control unit calibrated against a National Bureau of Standards certified thermometer. Only the interference optics were used for these experiments. The alignment procedure has been described previously (23, 24). Photographs were taken mainly at the two-third focus of the camera lens. Photographs were recorded on Kodak spectroscopic plates, emulsion type IIG, and developed with H.R.P. developer (Kodak). Exposure times of lo-15 min were necessary when using a Polaroid filter on top of the 77-A filter, with the limiting aperture of the interference optical system reduced to 1 mm, and with the AH-6 lamp voltage reduced to 90% of its usual value (25). High speed sedimentation equilibrium experiments were performed as described (6, 25) using six-

498

SZUCHET

AND

channel “Rexolite” centerpieces 12 mm thick and sapphire windows. All experiments were performed at 52,000 f 200 rpm and 20.0 + O.l”C. Equilibrium was attained in about 9 h for the 3 mm high solution columns used. In all cases, however, at least 24 h was allowed for approach to equilibrium. Each experiment with a protein solution was preceded and followed by a water blank, run in the same cell without disassembly. Initial protein concentrations were determined with a Zeiss spectrophotometer using an extinction coef?icient E&, = 0.96 at 278 nm (141, assuming no change of extinction coefficient in acid since knowledge of exact concentrations was not required. Concentrations as a function of position were calculated from fringe displacements using the value 0.1822 g/ml for the refractive increment of p-Lg at 546 nm in dilute salt solutions (26). Under the experimental conditions used, a l-mm fringe displacement corresponds to a concentration of 0.87, g/liter. The partial specific volume of p-LgA was assumed to be 0.751 ml/g (27). A value of 18,463 was assumed for the molecular weight of the 6-LgA monomer. This value was calculated from the amino acid sequence (21) and is believed to be more reliable than values obtained frcm amino acid composition or measured by other physical methods. Values of the densities and partial specific volumes for acetic acid solutions were obtained from Szuchet and Yphantis (6). The densities used for estimation of solvent densities and for our calculations of the partial specific volume of the aqueous acids were the data of Liideking (281, Drude (29), and Landolt (30) for propionic acid and the observations of Grindley and Bury (31) for butyric acid. Our estimates at 20°C of the solvent densities (including a small contribution from the 0.1% sucrose used to provide a stabilizing density gradient), the partial specific volumes of the acids and the buoyancy factors estimated for the various acids are presented in Table I. No correction was applied for the density increment of the protein itself. As before (61, the ionic strength (~1 of these organic acids was estimated as -(m,&.)1’2. In these experiments our estimates of m3 ranged from 0.0012 M for 0.1 M propionic acid to 0.0132 M for 10 M acetic acid. Data analysis. Two Fortran IV computer programs were used for the analysis and interpretation of data, “Biospin” (32-34) and “Nonlin” (35, 36). Biospin calculates various pointaverage molecular weight moments. From the behavior of these moments one may infer and then test a specific model. Nonlin, on the other hand, performs a least-squares fit of the data to an assumed model. These programs, therefore, offer alternative and complementary methods of analyzing equilibrium data. Typically, information from Biospin was used to suggest models and initial “guesses” to be tested with Nonlin.

YPHANTIS TABLE I SOLVENT DENSITIES AND PARTIAL SPECIFIC VOLUMES OF AQUEOUS ORGANIC ACIDS AT 20°C Acid

Acetic

Propionic

Butyric

Molarity (mobliter)

Density” (g/ml)

0, (ml/ g)

(l-O@)

0.1 1.0 10.0 0.1 1.0 10.0 0.1 1.0 10.0

0.9994 1.0072 1.0625 0.9993 1.0051 1.0241 0.9991 1.0029 0.9754

0.856 0.860 0.918 0.88, 0.90, 0.99, 0.94, 0.96, 1.036

0.145 0.134 0.025 0.116 0.091 -0.015 0.057 0.030 -0.011

LIIncludes a contribution presence of 0.1% sucrose.

(+0.00038 g/ml) for the

Specifically, Biospin calculates the four standard apparent point-average molecular weights (number, weight, z, and z + 1) as experimental observables; the odr) (24) at each appropriate observation radius, r. It also calculates seven “ideal” molecular weight momenta (uy’s) that are identically independent of the second virial coefficient for systems composed of a single molecular species or for reversibly associating systems (37). In addition, some of these “ideal” moments are also independent of the third and/or fourth virial coefficient. Thus, these “ideal” moments may, in principle, remove the effects of even severe nonideality arising from excluded volume and/or Donnan effects. Nonlin is an adaptation of the Newton-Raphson nonlinear least-square fitting algorithm (38) to sedimentation equilibrium data. [A similar program for analyzing temperature jump data as a sum of up to three exponential terms has been described by Johnson and Schuster (39)]. This program fits experimental data to models with one, two, or three molecular species of either undetermined or preassigned values of oi = [Mi(l-ir,p)oZ/RTl with up to four nonzero colligative virial coefficients. Rough estimates of initial ‘guesses” for the various parameters are required and the program refines these “guesses” to a specified precision (35). Lack of computer facilities should not be a deterrent to the application of the solvent systems described here even though we have used two complex computer programs for the analysis of the data presented. The degree of complexity of the analysis of data ultimately should depend on the type of information one wishes to extract from the system and on the accuracy of the available data. If the information sought is the molecular weight of the subunits and an estimate of the nonideality and homogeneity of the system, then the data can be analyzed adquately with a desk calculator. For example, a sim-

CENTRIFUGATION

OF LACTOGLOBULIN

ple, rapid procedure has been described (24) for estimating the point-weight average molecular weights. The usual plots (i.e., uw(r) vs C(F) for a number of initial loading concentrations) then can be made to detect heterogeneity. Since the systems under consideration are highly nonideal the more familiar plot of In c vs 13/2 should not be used. A useful description of how to handle sedimentation equilibrium data with a programmable desk calculator has been given by Aune and Timasheff (40). RESULTS

0.1 M Solutions

IN ACID

499

07.

06. e i OSb’ 04

03-

02

,

of Acids

The behavior of /3-LgA in 0.1 M acetic, propionic, and butyric acids is depicted in Fig. 1 where we present a graph of l/a,(r), the reciprocals of the apparent reduced point-weight average molecular weights, vs the point concentration, c(r)‘), for the three acids. Several features of Fig. 1 are noteworthy. First, the three curves, corresponding to three independent experiments in different acid systems, converge to give almost a single straight line in the limit for concentrations below approximately 1.2 mm of fringe displacement (

Equilibrium centrifugation of proteins in acidic solutions. Beta-lactoglobulin A in aqueous acetic, propionic, and butyric acids.

ARCHIVE8 AND OF BIOCHEMISTRY Equilibrium 173, 495-516 (1976) BIOPHYSICS Centrifugation Beta-Lactoglobulin of Proteins A in Aqueous SARA SZUC...
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