Protein-Ionic Detergent Interaction

VOLUME AND l~I,~~CTROI’HOI~ETIC CHANGES PRODUCED B,I- SODIURI DODECYL SULFATE REACTION WITH BOVIKE SERUnI AI,BUnIIS*

(Received for publication, February 22, 1972)

SAM KATZ, I\IARY E. SHAW, SEIAWX CHILLAG, AND JANE El. ~IILLER

From, the Biochemistry Depwtmeut, West Virginia University Jledical C’eutel~ , ll/organtowtr, West Virginia 26506

SUMMARY

The volume effects produced by the reaction of sodium dodecyl sulfate with bovine serum albumin were determined dilatometrically at 30.0 + 0.001”. The calculated volume isotherms indicate the operation of at least two major proc- esses: (a) a volume decrease which is caused by a high affin- ity protein-detergent interaction and occurs at detergent concentrations ranging from 0.02 to 0.08 M; and (b) a volume increase which occurs at SDS concentrations >0.08 M and is attributed to detergent interaction with the structurally altered protein. At detergent concentrations less than 0.02 M either positive or negative volume changes are produced depending on the type and ionic charge of the protein. The effect of pH was investigated by comparing the volume iso- therms produced by albumin at pH 5.1 and at pH 8; the negatively charged albumin exhibited a more pronounced volume decrease compared with the isoionic albumin. The volume isotherms are a unique function of the composition, structural organization, and charge of the protein.

Acrylamide gel electrophoretic studies of these systems reveal the generation of at least two categories of protein- detergent complexes. At low ratios of sodium dodecyl sul- fate (SDS) to protein, there is a progressive increase of the mobility of protein complexes with increasing anion concen- tration. At detergent concentration 20.02 M another com- ponent is formed whose mobility is substantially faster than that observed at lower concentrations. At SDS concentra- tions LO.08 M this is the only component present; the mo- bility of this component is invariant in the concentration region of 0.02 to 0.5 M SDS.

Even though an estensive literature esists pertaining to the reaction of proteins wit,11 ionic detergents (1, 2), there is a paucity of information relative to the volume effects produced by these processes. This report deals with the volume change produced

* This research was supported in part by United States Public Health Service, National Heart and Lung Institute Grant HE12953. ,4 portion of this paper was presented at the 162nd National Meeting of the American Chemical Society at Washing- ton, L). C., September, 1971.

by the interaction of sodium dodecgl sulfate with bovine serum albumin, a single-chain globular protein, molecular weight about 66,000. Esperiments were performed with IBA’ whose initial pH was 5.1, essentially isoiollic, and at $1 8.0. The volume effects produced by increasing the ratio of SDS to protein appear to be the resultant of at least two major processes: (a) a volume decrease ascribable to the binding of SIX to the high affinity sites of protein and (b) a volume increase which occurs when the ratio of SI)S to protein exceeds a given point generating a drastic structural change of the protein. 1X%4 initially at a pH near its isoionic point exhibited a sharp volume increase when 2c/; protein was exposed to 0.005 to 0.02 $1 SIB; however, BSA initially adjusted to pH 8 did not manifest this phenomenon.

Acrylamide gel electrophoretic analysis of these systems re- vealed that increasing SDS concentration from 0.001 to 0.07 M, in systems containing 2yT protein, caused a progressive illrrease of the electrophoretic mobility of the protein-SDS complexes formed. At SDS concentrations 20.08 M SDS, only one c'oIn-

ples was demonstrable; the mobility of this comples was sub- stant.ially faster than that, associated with the rategory of rom- pleses present at loner SI)S concentrations. This transition zone coincided with region where the inflection point occurred in volume isotherms calculated from the dilatometric data.

EXPERIMESTAL PROCEDURES

Xetlr.ods-The volume changes were determilled with l’efioll- sheathed Linderst,r#m-Lang type dilatometers consisting of a 10.~1 capillary which could be read to 0.01 ~1 and of a bifurcated reaction vessel (3). The experiments were performed at 30.0” f 0.001”; the methodology has been described previously (4, 5).

The AV d&a (AV is the volume change in microliters produced by mising the txo components of a system) are the steady state values measured 5 to 15 min after misiug; no kinetic effects were observed during the 100 min after misiug. The mixing protocols were as follows. (a) For the 5 :5 program 5 ml of SDS were added to 5 ml of aqueous phase: the SI)S collcentra- tion after mixing ranged from 0.01 to 0.10 M.~ (b) For the 8:2 program 8 ml of SI )S were mised with 2 ml of aqueous solution; the SIM concentration ranged from 0.005 to 0.50 IRI SDS. The

1 The follo\vnrg >ibbrevintions are llsed: BSA, bovine serum albumin; SDS, sodium dodecyl sulfate.

2 The SDS concentration stated in this text indicates that total SDS concentration after mixing; in water, the SDS coexists as individual molecules and micelles. In the presence of protein, a sizable amormt of t,he SDS is bound to the protein (I, 2).

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concentration of protein after mixing was 2.07;, f 0.150/,, w/v. The following systems were studied: BSX, essentially isoionic, pH 5.1, and BSA, adjusted to pH 8 with cold 0.1 M NaOH.

The kinetics and mechanisms of the reactions were studied by acrylamide gel electrophoresis employing procedures described previously (5, 6). The experimental design simulated the dilatometric experiments; 5- to lo-p1 samples were used for analysis. The albumin systems were resolved with Peacock’s buffer (7); this buffer does not include SDS. Electrophoresis was conducted at 250 volts, electrolyte temperature was main- tained at 15” f 2”, and the run duration was 2+ hours.

Reversibility experiments were performed with 2o/, BSA ex- posed to 0.1 M and 0.2 M SDS for 2 hours. One milliliter of this solution was added to 2 g of Dowex 1-1X4-100, chloride form, and agitated for 30 set with a Vortex stirrer. After 10 min of equilibration the mixture was centrifuged. The supernatant was subjected to thin film dialysis against glass-distilled water for 4 hours (8). The ratio of inner to outer solutions was 1:400; the outer solution was changed at 3 hour intervals. The dialysis rate was monitored with a Radiometer type CDM3 conductivity meter. The pH was measured with a Radiometer pH meter model 26 calibrated with two Harleco standard buffers; solutions containing SDS required 3 to 5 min to reach steady state.

SDS analysis was performed with a modification (9) of the Mukerjee procedure (10). Protein concentration was deter- mined with a Beckman DU-2 spectrophotometer employing values of E::m of 6.67 at 279 nm for BSA (11).

dl&k&-BSA, crystallized, was purchased from Metrix Division of Armour Pharmaceutical Co.; sodium dodecyl sulfate and tris(hydroxymethy1) amino methane w-ere products of Sigma Chemical Co. The reagents for acrylamide gel electrophoresis were from Fisher Chemical Co. The remainder of the analytical grade reagents were obtained from Mallinckrodt Co.

RESULTS

A prerequisite for this study was the determination of the AV produced by the dilution of SDS by water. The volume changes for the 5 :5 and 8 :2 protocols are summarized in Fig. 1. The AV isotherm produced by the 5:5 protocol decreases in a nearly linear manner in the region of 0.01 to 0.10 JI SDS; at the con-

SDS x 102M

O.Ob IO 20 30 40 50

I

0 I 2 3 4 5 6 7 6 9 IO

SDS x lo%

FIG. 1. Volume changes produced by mixing SDS with water as a function of volume and concentration. O- - -0, volume changes in microliters produced when 8 ml of SDS are mixed with 2 ml of water; SDS concentration, after mixing, is indicated on the top abscissa. +w, volume change produced when 5 ml of SDS are mixed with 5 ml of water; SDS concentration is indicated on the bottom abscissa. Temperature is 30.0” & 0.001”.

centration extremes there is an abrupt change of slope. The isotherm produced by the 8:2 protocol, encompassing a concen- tration range of 0.01 to 0.50 M SDS, exhibits a sigmoidal rela- tionship with respect to SDS with values for AV decreasiilg from -0.02 ~1 at 0.01 M SDS to -0.57 ~1 at 0.50 RI SDS.

The volume changes produced by the reaction of SDS with proteiu is a function of the protein type, pH, concentration, type, and volumes of the reactants. The considerable influence of t,he mixing protocol is illustrated by reference to the data from a single experiment summarized in Table I. The volume changes produced by the reaction of SDS with 20; USA, pH 5.1, obey a sigmoidal relationship with increasing detergent concentration

(Columns 2 and 5, Table I). When the 8 :2 mixing protocol is employed, a AV of 0.22 ~1 is obtained at 0.01 M SDS; at 0.02 RI

SDS, AV is negative, the value being -0.10 ~1; at 0.08 M SDS a minimum of -0.72 ~1 is obtained. At SDS concentmtions >0.08 M the magnitude of AV decreases yielding a value for AV of -0.01 ~1 at 0.5 M SDS. Sirnilar effects are observed when the 5 : 5 mixing protocol is used. This protocol was not eniployed at SDS concentrations >O.lO M because of the large water-SDS interaction term (see Fig. 1). BS4, adjusted to pH 8, produced volume effects which were more negative than that produced by isoionic BSA. The values for the 8:2 mixing protocol were -0.12, -0.43, -0.88, and - .65 ~1, at 0.01,0.02, 0.08, and 0.5 1\1

SDS. The difference between the isoionic and the alkaline forms of BSA is especially pronounced at SDS concentrations 20.1 hr. Isoionic BSA exhibits a pronounced rise of slope with increasing SDS concentration while the alkaline form of BSA does not.

The possible loss of SDS by transfer from the aqueous to the heptane phase upon mixing was investigated. Partition studies simulating the dilatometric experiments were performed employ- ing a ratio of 3 volumes of heptane to 1 volume of water. The systems were vigorously mixed for 5 min and allowed to equili- brate for 3 hours. There was an insignificant decrease of SDS concentration in the water phase at 0.02 M SDS; however, at 0.1 and 0.5 M SDS about a 5yG reduction of concent’ration was noted. The transfer of SDS during dilatometry will be minor

TABLE I ATi produced by mixing SDS with proteins

For the 8:2 protocol 8 ml of SDS were added to 2 ml of 10% pro- tein; for the 5:5 protocol 5 ml of SDS were added to 3 ml of 47; protein. For experimental details see text (temperatlrre was 30.0”).

Total concentration

SDS X lo*

1

2 4 6

7 8

10

15 20 30 40

50

AP

8: 2 Protocol 5:s Protocol

lo”w”,“,t =A, PHI 8 lIr0

0.22 -0.12

-0.10 -0.43 -0.38 -o.c58 -0.60 -0.70

-0.72 -0.88 -0.71 -0.84

-0.55 -0.75 -0.36 -0.71 -0.25 -0.68

-0.12 -0.67 -0.01 -0.65

-0.02

-0.04 -0.08 -0.09

-0.09 -0.10 -6.11

-0.15 -0.16 -0.22

-0.33 -0.57

-0.09 -0.12 -0.22

-0.22 -0.49 -0.26

-0.54 -0.74 -0.29

-0.79 -0.91 -0.31

-0.86 -0.97 -0 .32 -0.92 -1.07 -0.33 -0.99 -1.08 -0.35

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5230

FIG. 2. Acrylamide gel electrophoresis of BSA exposed to SDS. The total concentration of SDS, expressed as molarity, is given on the bottom of the photograph. A 2% solution of protein was exposed to the detergent for 5 hours at 30” before electrophoresis. Elec- trophoresis was performed in 7.5% acrylamide at 250 volts for 24 hours at pH 9.1 (see text for specific details).

since vigorous mixing was avoided; furthermore, the contact area between the phases was about 10% that used in the parti- tion experiments.

Electrophoretic analysis provided additional insight with respect to the mechanism of binding. BSA exhibited a nearly monotonic increase of electrophoretic mobility as the SDS con- centration increased from 0.001 to 0.06 M (Fig. 2). At an SDS concentration of >0.02 M a second fraction appeared whose mobility was substantially faster than either BSA or the slower BSA-SDS complexes. At SDS concentrations >0.08 M this was the only complex present; the mobility of this fraction is virtually concentration independent. The protein-SDS complexes re- solved by this technique must be extremely stable since 5 to 10 ~1 of protein-SDS mixture is introduced into a large excess of SDS- free electrolyte and then subjected to electrophoresis.

Reversibility experiments were performed with 2% BSA ex- posed to 0.1 M SDS for 1 hour. Attempts to remove SDS by thin film dialysis (8) were not satisfactory since about 40% of the detergent remained after 24 hours of dialysis. Renaturation was achieved by batch treatment with a anion exchange resin which removed 90% of the SDS; the remainder of the detergent was removed by thin film dialysis for 4 hours. The renatured protein was identical with the starting materials with respect to solubil- ity, electrophoretic properties, and reactivity to urea and SDS.

DISCUSSION

The volume effects produced by the addition of SDS to protein originates primarily from two factors, SDS-protein interaction

and SDS-water interaction. The magnitude of the initial effect can be determined from the following relationship which we define as the 6V function

sv = AVzd - Al/‘~d . 1o5

(1) w

where AVzd is the volume change produced when a defined volume and concentration of protein, Component 2, is mixed with a defined volume and concentration of detergent, Component d; AVIS is the volume change produced by the identical system, except that the protein is omitted; w2 is the protein weight in grams. This is multiplied by lo5 to convert this parameter to lo5 g of protein. The protein-water dilution term is not in- corporated in this equation since its partial molar volume con- tribution is invariant in the concentration ranges employed (4, 6). This definition of 6V implies that the magnitude of water- SDS interaction in a system containing protein is presumed to be identical with that of a protein-free system; the implication is that the protein bound SDS produced the same effect on water structure as free SDS. This simplification results in an over- correction; the uncertainty introduced by this assumption will be discussed subsequently. Another source of error is the approxi- mation that the volume of water in a system containing protein is consid,ered as being identical with that of a protein-free solu- tion. The effective water volume in a protein solution can be determined by subtracting the volume occupied by the protein (the product of the protein’s weight by its partial specific volume)

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8’

I I IO 20 30 40 50

SDS MOLARITY x IO’

P‘Ic;. 3. The 611 isotherms produced by the reaction of BSA Cth SDS. -, isotherm for BSA, pH 3; 0, values obtained by using the 8:2 mixing protocol; l , values determined from the 5:G mixing protocol; - - -, corresponding isotherm for BSA adjusted to pH 8; the experimental points were omitted to minimize con- f[lsioll. TemperatLtre \vas 30.0”.

from the total volume. The corrected volumes for the 8:2 and 5:s mixing systems were 1.85 and 4.85 ml, respectively. When these volumes of water were employed in SDS-water mixing experiments (see Fig. I), it was found that the differences be- tween these two sets of data were 50.04 ~1 for concentrations ranging from 0.01 to 0.3 M SDS. At SDS concentrations >0.3 M

the difference was larger and conceivably introduces a potential source of uncertaint’y.

The 6V isotherms functional dependence on SDS concentration is determined primarily by the protein’s composition, conforma- tion, and charge. The slope and sign of the 68 isotherms change drastically as a function of SDS concentrations, namely in the regions encompassed by 0.005 to 0.02, 0.02 to 0.09, and 0.09 to 0.5 II. The most disparate effects occur in the region where the SDS concentration is <0.02 M. For example BSA, initial pH 5.1, eshibits a volume rise in this region as reflected by 6V of 120 ml/10 g of protein at 0.01 M SDS; whereas BSA, initial pH 8, produced a 6V of -35 ml/lo5 g of protein at this concentration (Fig. 3). This difference between the volume effects which occurs as a function of pH at low SDS concentrations may be a consequence of the “albumin neutral transition,” i.e. the struc- tural transition produced when the pl-I is raised from 5 to 9. Evidence for this subtle conformational change has been ob- tailred by calcium ion binding (12), optical rotatory dispersion (I 1)) and, recently, by titratiou studies (13). The positive value for 6V for isoionic albumin is explicable in terms of SDS binding to high affinity sites in the protein.3 This binding may involve all electrostatic interaction between the anionic detergent with positively charged groups thereby displacing water of electrostriction and thus producing a positive volume rise (14).

I,inderstr$m-Lang and Jacobsen (15) have compiled convincing

evidence that the interaction of positively and negatively charged

zf The volume rise noted for interaction of isoionic BSA with low concentrations of SDS, <0.02 M, may be considered as a mani- festation of a neutralization reaction since a positive volume rise was observed. It also can be argued that when the pH of the system is raised to >pH 8 the buffering action of the protein would mask this effect. This argllment can not be refuted with the data for P&4; however, our unpublished data for isoionic myoglobin reveals a larger initial SV values and lower pH changes in this region thus arglling against a contrib\ltion due to neutralization.

residues produce volume increases ranging from 10 to 20 ml per mole of reacting ion pair. There are several factors which mitigate against the alkaline form of 13%~ exhibiting this effect at low SDS concent’rations, namely (a) the population of the reactive posit’ive sites decrease with increasing pH, (b) geometri- cal considerations associated with the neutral transition form of BSA do not favor this interaction, i.e. binding at low SDS con- centrations decreases substantially as the pH of the system increases from 6 to 8 (16, 17). The influence of SDS on this phenomenon attributable to micelle dissociation is minimal. It is recognized t’hat the critical micelle concentration and t’he num- ber of molecules associated in the micelle vary as a function of salt concentration (18, 19), temperature (20), solvents (al), etc. At low SDS concentrations, <0.02 M, the binding of SDS to the protein reduces (dilutes) the SDS concentration and this can cause volume effects. Reference to Table I and Fig. 1 will reveal that the magnitude of this effect for the 8:2 mixing protocol would amount to <0.02 ~1; consequently this would constitute a second order effect.

At SDS concentrations >0.02 RI the values and slope for the 611 isotherms are negat,ive; these systems upon electrophoresix are characterized by complexes which exhibit progressively more negative electrophoretic mobility as the SDS concentration in- creases. The mobility data is indicative of an increased electro- static charge density for the protein-SDS complexes; the volume decrease is a measure of the electrostriction effect (14). &4n estimate of the volume (sontraction produced by these charge effects call be determined by t,he use of the Drude-Nernst equa- tion (22,23). This states that the volume change, AV, produced by the introduction of a sphere of radius, r, w&h an electrical charge, eZ, distributed uniformly over the surface into a medium is given by the following relationship

-p(eZPV dD Av=---------.- 2rD2 dV

where V is the volume, p the compressibility, and L> the dielectric constant of the medium. Consequently anion binding which causes an increased electrostatic charge will produce a geometric volume decrease since this constitutes an exponential term in the above equation. It is recognized that other factors in Equa- tion 2 are altered by the introduction of SDS; however, their contribution will not be as significant as the charge effect. Kauz- mann and colleagues (24) determined that the orientation and location of Dhe charged groups in a polyfunctional compound has a profound influence on electrostriction. For example, the volume changes produced by the protonation of the salts of doubly charged dicarboxylic acids whose prototropic groups are in close juxtaposition thus establishing overlapping electrostatic fields produced an enhanced electrostriction effect in accord with t)he Drude-Eernst equation. However, where the carboxylate groups were widely separated, the volume changes simulated those produced by isolated monocarbosglic acid functions. Similarly, Glueckauf (25) confirmed that the hydration volumes of ions are influenced to a considerable degree by geometrical considerations.

The 6V isotherms for the proteins studied exhibit a minimum at about 0.08 M SI>S; at higher detergent concentrations the isotherms describe a sigmoidal curve with a positive slope. This inflection is indicative of the occurrence of a substantial structural transition for these proteins. This hypothesis is in accord with similar interpretations based on spectral, optical rotation, and

viscosity &dies of albumin (11, 26). Another manifestation of

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this structural change is the large increase of electrophoretic mobility of the protein-SDS complexes at 20.06 RI SDS concen- tration, Fig. 2. This volume increase may be the result from the operation of several factors. The most significant one being caused by the expansion and extension of the protein’s surface (26), thereby reducing the localized clustering of the protein- bound SDS molecules, thus decreasing the degree of overlapping electrostatic field contribution wit’11 a consequent decrease of the electrostriction effect. Furthermore, the loosening of the pro- tein’s structure can increase the protein’s void volume, regions inaccessible to the solvent, aud thus cause a physical expansion of the system (4, 27).

Another factor which may influence these volume effects is the pH shift which results from ion binding. The relationship betweeu ion binding and the resultant pH shift is as follows

ApH = 0.87wA; = 0.87~8 (3)

where 0 is the rnean number of ions bound per protein molecule, AZ is the mean increase of charge due to binding, and w is the electrostatic interaction term (28). A restriction implicit in the derivation of this equation is that the number of protons bound to the protein is not affected by the binding process. Cassel and Steinhardt (29), in a definitive study, concluded that when the protein concentration was > I”/;, this condition was met. Since 2Ljb protein concentrations were employed for these studies the volume effects attributable to either protonation and neutraliza- tion processes should be of secondary importance (27, 30). An- other approach to establish the possible effect of pH changes on volume effects is as follows. The pH of isoionic BSA exposed to 0, 0.02, 0.08, and 0.5 M SDS was 5.15, 7.0, 7.5, and 7.8 units, respectively. I f these pH changes were associated with the neutralization of the nitrogenous acidic functions in BSA, there would be successive volume increases of 275, 50, and 75 rnl/lOj g of BSA, respectively (30). I f the volume effects were produced by proton transfer from nitrogenous acids, the consequent volume increases would be 10 to 204’/, that produced by neutralization processes (27, 30, 31). Another analysis is provided by inspect- ing the volume changes produced by the reaction of SDS with BSA, adjusted to pH 8. The pH of the system increased from 8 to 8.8 at 0.02 M SDS and then plateaued at 9.1 at SDS concentra- tions 20.04 M. I f this pH shift were caused by the neutralization of nitrogenous bases there would be a volume rise of about 200 ml/lo” g of BSA at 0.04 M BSA; however, the experimental value was -240 ml per 10”. At SDS concentrations 20.04 hf there would be no volume changes whatsoever. The lack of cor- respondence between the observed and calculated values indicate that neutralization processes are of minor significance.

The 6V values calculated from Equation 1 are influenced by the criteria for selecting the AVld term. We assume that the AVld term is a function of the total SDS concentration, i.e. the magnitude of the water-SDS interaction is determined by the total SDS present this gives equal weight to the contribution of the protein-bound SDS as it does to the free SDS. Let us con sider the other boundary condition, namely that AVld is deter- mined only by the concentration of free SDS. Before making a detailed analysis, inspection of the data in Table I and Fig. 1 reveal that the magnitude of the water-SDS correction for the systems included in the 8 :2 mixing protocol will not introduce a rnajor error at SDS concentration <0.15 M; however, for the 5:5 mixing protocol this factor can assume serious proportions at all levels of concentrations. Consider the system containing iso- ionic BSA and 0.02 M SDS, 8:2 mixing protocol; if we assume that 5OGj, of the SDS were protein-bound then AVld would be

reduced from -0.04 ~1 to -0.02 ~1 (see Table I, Fig. 1). The resulting 6V would be -40 ml/lo5 g of USA instead of -30 ml/lo5 g of BSB. If we consider the same system, but employ the 5:5 mixing protocol, the value of 6Vld would decrease from -0.26 to -0.22 ~.d, changing the value of 6V from -20 ml/lOj g of BSA to -0 ml/lo” g of 1%~. For the 0.10 hf SDS system, employing a 8:2 mixing protocol, and assuming 500/; bindirig, a corrected AVld value of -0.09 ~1 would be obtained compared with -0.10 ~1 by the previous approach. This would yield a 6V of -310 ml/lo5 g of BSh compared with 300 ml/lOj g of BSA originally calculated. The same system, 5:5 mixing pro- tocol, would give a corrected AVld of -0.30 ~.d compared lvith -0.35 ~1; the resulting value for 6V would be -345 ml/lo5 g of BSA compared with -320 ml/lo5 g of USA. If one considers the system containing 0.5 hf SDS and if we assume that the pro- tein is saturated with SDS, i.e. 1.4 g of SDS complexed per g of BSA (32, 33), the value for AVld would decrease from -0.57 to -0.30 ~1. The value 6V would decrease from 280 ml/lo5 g of BSA to 145 ml/lo5 g of BSA, nearly a 100% variation. It can be concluded that the 8V values determined from the 5:5 mixing protocol, encompassing 0.02 to 0.10 M SDS or from the 8:2 mixing protocol, 0.01 to 0.3 h1 SDS, are not influenced materially by the criteria for selecting AVld. However, when the SDS conceutra- tions is >0.3 M, the basis for selection of this term can introduce a serious error. The above calculations are for isoionic BSA; however, similar consider&ions are applicable to the other systems studied.

At present it is not feasible to relate the 68 function with the amount of SDS bound to the protein. The volume effects reached steady state within 5 to 10 nun aft,er mixing; the t)ime required for binding equilibrium t’o be achieved is still not es- tablished. SDS binding data, determined by dialysis, requires an extended period of time before equilibrium is achieved (34). Therefore, additional kinetic and thermodynamic data are required in order to make this correlation (33).

One concludes that the 6V isotherms are a function not only of the protein’s composition and three-dimensional structure, but also of its initial charge distribution. The unique character of the ascending portion of the 6V isotherms suggests that the SDS denatured protein retains sufficient structural integrity to pro- duce volume effects characteristic of a given protein. The analy- ses of these isotherms may provide a new approach for determin ing the degree of structural integrity retained by proteins upon exposure to denaturing agents.

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Sam Katz, Mary E. Shaw, Shawn Chillag and Jane E. MillerWITH BOVINE SERUM ALBUMIN

CHANGES PRODUCED BY SODIUM DODECYL SULFATE REACTION Protein-Ionic Detergent Interaction: VOLUME AND ELECTROPHORETIC

1972, 247:5228-5233.J. Biol. Chem. 

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