Oct., 1958
BINDINGOF SODIUM DODECYL SULFATE BY SERUM ALBUMIN
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THE BINDING OF SODIUM DODECYL SULFATE BY SERUM ALBUMIN I N THE ABSENCE OF ADDED ELECTROLYTE BY GEORGESTRAUSS~ AND ULRICHP. STRAUSS Ralph G. Wright Laboratory, School of Chemistry, Rutgers University, New Brunswick, New Jersey Received M a y by, 1968
An electrical transference method was used to study the interaction between bovine serum albumin and sodium dodecyl M , the average number of detergent sulfate in the absence of added electrolyte. At protein concentrations of 3 and 6 X anions bound per protein molecule was independent of the protein concentration and increased with the concentration of free detergent, reaching approximately 160 a t the critical micelle concentration (c.m.c.) of the detergent. This number also was obtained independently from shifts in the apparent 0.m.c. as determined by specific conductance measurements. The interaction was shown to be reversible by conductance and viscosity methods. The average fraction of sodium ions dissociated from the protein-detergent complex decreased as more detergent became bound and reached 0.4 a t the c.m.c. cm.* volt-' set.-* when the number of bound detergent anions The mobility of this complex increased from 31 to 34 X increased from 50 to 160. Serum albumin did not bind aliphatic hydrocarbons which indicates that it lacks large hydrocarbon regions.
Introduction Serum albumin combines with sodium dodecyl sulfate to form complexes of varying stability. The reaction has been studied by several methods including v i ~ c o m e t r y , ~electrophoresis16 -~ equilibrium dialysis,'J dye displacementg and precipitation.2J0 Qualitatively it has been concluded that the protein unfolds as more detergent becomes bound, resulting-at least during the early stages of binding-in the exposure of additional binding sites." Moreover, the behavior of serum albumin is strongly dependent on its ionic environment. Most of the investigations cited above were carried out in the presence of buffers or other supporting electrolytes. Since serum albumin binds small anionsI2 which consequently compete with detergent ions, it was desirable to follow the proteindetergent reaction in the absence of any extraneous electrolyte. An electrical transference method, developed by Wall, et aE.,lafor binding studies on macromolecules and small ions, appeared suitable for the present purpose and was adapted for use with the serum albumin-sodium dodecyl sulfate system. As will be shown in the next section, the average number of detergent anions bound per protein molecule, the mobility of the proteindetergent complex and the average fraction of sodium counterions dissociated from this complex can all be determined by the transference method. (1) Colgate Post-Doctoral Fellow 1955-1957. (2) F. W. P u t n a m and H. Neurath, J . A m . Chem. Soc., 66, 692 (1944). (3) H.Neurath and F. W. Putnam, J. B i d . Chem., 160, 397 (1945). (4) E. L. Duggan and J. M. Luck, ibid., 172,205 (1948). (5) B. 8. Harrap and J. H. Schulman, Disc. Faradau Soc., 13, 197 (1953). (6) F. W. P u t n a m and H. Neurath, J . B i d . Chem., 169, 195 (1945). (7) F.Karush and 111. Sonenberg, J . A m . Chem. Soc., 71,1369 (1949). (8) M. J. Pallanach and D. R. Brigga, ibid., 76, 1396 (1954). (9) I. M. IClotx, H. Triwush and F. M. Walker, ibid., 70, 2935 (1948). (10) K.G. A. Pankhurst and R. C. M. Smith, Trans. Faraday Soc., 40, 565 (1944). (11) For reviews of binding studies see (a) F. W.Putnam, Advances i n Protein Chem.. 4, 80 (1948); (b) M. E. L. McBain and E. Hutchinson, "Solubilization," Academic Press, New York, N. Y.,1955. (12) G. Scatchard, I. H. Scheinberg and 8. H. Armstrong, J. A m . Chem. Soc., 72, 535, 540 (1950). (13) J. R. Huizenga, P. F. Grieger and F. T. Wall, ibid.. 79, 2636 (1950). Recently an article appeared in which essentially the same
method was used to study the binding of small inorganic ion8 by serum albumin [R. II. Doremus and P. Johnson, THISJOURNAL, 62, 203
(1958)1.
Theory and Method Calculation of the Number of Bound Detergent Anions.-If an electric current is passed through a protein-detergent solution in a Hittorf-type twocompartment transference cell, some of the free detergent anions (D-) and some of the negatively charged protein-detergent complex will migrate from the cathode to the anode Compartment while some of the free Na+ ions will travel in the opposite direction. If an average of T D- ions and m Na+ ions are bound to the protein molecule, the total number of moles of D- and of Na+ moving from one compartment to the other will each be made up of the amounts traveling in the free and in the bound state. Designating these total amounts as AD and ANa, respectively, and t.he amount of protein migrating as AP, in moles per faraday, we have
+
AD = t ~ - rAP ANa = t N s t - mAP
(1) (2)
where t ~ and - t N a + are the transference numbers of free D- and Na+, respectively. According to the customary definition of the transference number of an ion, t D - is given by where AD- is the equivalent conductance of the free D- ion (cm.2 ohm-' equiv.-l), d and p are the stoichiometric concentrations of detergent and protein (moles per liter) and K is the specific conductance (ohm-' cm.-I) of the solution due to all ions other than H+ and OH-. The contribution of the latter ions to the tot,al conductance was found to be negligible in the solutions investigated'4 so that the value of K , as defined here, may be taken as that of the measured total conductance. By combining equations 1 and 3, and rearranging (4)
so that r can be calculated from the measured values of AD, AP and K of a given solution. In practice, the change in the amount of Na+ ion, ANa, can be determined more conveniently than AD, and an expression relating ANa t o AD is desirable. If the (14) The specific conductance due to H + and O H - is given by (CE+XH' COH-XOH-)/~OOO.In our solutions this amounted to 0.2% or less of the total conductance.
+
GEORGE STRAWSAND ULRICHP. STRAUSS
1322
charge of the protein is due solely to the D- and N a + ions bound to it, it can be shown that AD = 1 - ANa
(5)
as required by the condition of electroneutrality.l5 The expression for r now becomes
Mobility of the Protein-Detergent Complex.This quantity may be calculated from transference data as follows. The transference number of the protein-detergent complex is given by (7)
where 5 is the faraday, z is the net charge of the complex and uc its mobility volt-l sec.-l). Consequently
Effective Degree of Dissociation of the Complex. we define the effective degree of dissociation of Naf ions from the protein-detergent complex, a,as 1 - (m/r), then equation 2 may be rewritten as
-If
ANa =
- r(1 - a)AP
(9)
The Concentration of free Na+ ions in solution is d - pr(1 - a) moles per liter so that the transference number tNa + is given by By combining equations 9 and 10 we find that
Equivalent Ionic Conductances.-The preceding equations require a knowledge of the equivalent ionic conductance h N a + and AD- at various ionic strengths. The values of AN^ t were taken from the literature’@while AD- was calculated from the experimentally determined equivalent conductance of sodium dodecyl sulfate solutions a t various concentrations and the published ANa + values. The‘ ionic strength of each protein-detergent solution which was required in order to assign proper values to the ionic conductances was taken to be equal t o the total detergent concentration. The presence of protein was ignored, for the reasons given by Tanford. l7 Experimental Materials.-Crystallized bovine serum albumin was obtained from Armour Laboratories. It was freed of fatty acids by extraction with acetic acid-isooctane as described by Goodman,’* and lyophilized. A 0.5% solution of this (15) When protein, originally at the isoelectric point, binds detergent, it becomes negatively charged. As a result it attracts H + ions as shown by the observed increase in pH. In this case equation 5 becomes A D = 1 ANa hAP h+, where h is the average number of H + ions bound by the protein and t ~ is+ the transference number of free H+. In our solutions, whose pH ranged from 6.0 to 7.1, the combined last two terms in the above equations amounted to not more than 0.03. This was less than the experimental error and so was neglected. (16) I€. S. Harned and B. B. Owen, “The Physical Chemistry of Electrolytic Solutions,” Reinhold Publ. Corp., New York, N . Y., 1943. (17) C. Tanford and J. G. Buzzell, THISJOURNAL,60, 225 (1956). (18) Goodman states that his method reduced the fatty acid content of serum albumin to 0.02 mole fatty acid per mole protein while
-
+
-
Vol. 62
albumin in water had a specific conductivity of 4 X 10-8 ohm-’ cm.-l. Pure sodium dodecyl sulfate was provided by the ColgatePalmolive Company. It had been synthesized19 from carefully purified materials and was substantially free of lauryl alcohol as shown by the absence of a minimum in the surface tension us. concentration curve. Conductivity water was repared by passing distilled water throu h a mixed-bed hmberlite MB-1) ion-exchange column. The specific conductivity did not exceed 1 X 10-6 ohms-1 crn.-I. Transference Measurements.-A cell having two compartments separated by a sintered glass disk, as described by Wall, et u Z . , ~ . was used. The electrodes consisted of platinum wire spirals. The total amount of electricity was determined by assing a regulated current for a known length of time. &ese measurements were confirmed by a hydrogen ion coulometer of the type described by Hoyer” which was placed in series with the transference cell. Currents of 1 to 3 milliamperes were used. The number of faradays passed ranged from 1 to 10 X 10-6 and were measured to within 1%. After each experiment the solutions were drained from each compartment, weighed and analyzed for total serum albumin and total sodium content. The original solution was similarly analyzed. Analyses.-Serum albumin was determined by measuring the light absorption a t 278 ma in a Beckman DU spectrophotometer. The absorbancy index at this wave length IS virtually independent of H changes between pH 6 and 7.1 and is not affected by t$e presence of detergent. Sodium was determined by use of a Perkin-Elmer Model 146 flame photometer, using the internal standard method. Since concentration changes rather than absolute concentrations were of interest in both these analyses, the solutions from the two cell compartments were compared with standard solutions: For each experiment a protein-detergent stock solution was prepared, portions of which were diluted to 90 and 80% of the original concentration. Part of the ‘‘90%” solution was used for the transference experiment while the “100,” “90” and “80%” solutions served as standards which bracketed the unknowns in the analyses. This precaution was especially necessary for the flame photometry because the presence of detergent somewhat affected the reading of the instrument. The transference runs were arranged so that not more than 8% of the total detergent or protein present migrated from one compartment to the other. The concentration changes could be determined to within 1% of their actual values. From these and the weights of the solutions from each compartment, ANa and A P were calculated. Conductance.-A conductivity bridge of the design of Edelson and Fuossa*was used. The cells had bright platinum electrodes and cell constants of 0.9030 and 0.8585 which were determined by calibration with KCl solutions.** Polarization errors were eliminated by extrapolating the readings obtained at 2000, 3000 and 4000 cycles to infinite All measurements were made a t frequency ( R us. f-’/z). 25”. Viscosity.-A Bingham2a viscometer having a ( p t ) ~constant for water of 8082 g. seconds per cm.* a t 25” was used.
Results and Discussion Number of Detergent Anions Bound.-Transference measurements were made a t 25” on a number of solutions containing protein and detergent. In the main series of experiments the protein concentration was kept a t 3 X M (assuming a mol. wt. of 69,000) while the detergent concentration was varied from 2 to 14 X 10-aM. A few purification by ion-exchange resins was not always so effective ID. S. Goodman, Science, 126, 1296 (1957)l. (19) E. E. Dreger, G. I. Keim, G . D. Miles, L. Shedlovsky and J . Ross, Ind. Eng. Chem., 86, 610 (1944). 60, 372 (1956). (20) H. W. Hoyer, THISJOURNAL, (21) D. Edelson and R . M. Fuoss, J . Chem. Educ., 27, 610 (1950). (22) G. Jones and B. C. Bradshaw, J . Am. Chem. SOC.,66, 1780 (1933). (23) E. C. Bingham, “Fluidity and Plasticity,” McGraw-Hill Book Co., New York, N . Y., 1922.
BINDING OF
Oct., 1958
SODIUM
DODECYL SULFATE RY
SERUM ALBUMIN
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ti 0 z U
zl
3
n
z
0 0
0 k
0
W 0.
cn
0
MOLARITY OF FREE DETERGENT x 10'.
Fig. 1.-Effect of free detergent concentration on the average number of detergent anions ( T ) bound per serum albumin molecule; results from transference: 0 , 3 x lo-' M protein; 0 , 6 X 10-6 M protein. Results from conductance a t c.m.c.: 0 , 3 X 10-6 M protein; a, 6 X loe6 M protein. The points represent average values, the length of the sloping lines the extent of experimental uncer- -, data of Pallansch and Briggss from equilibtainty. rium dialysis.
4 8 12 16 20 24 MOLARITY OF TOTAL DETERGENT x io3.
Fig. 2.-Specific conductance of sodium dodecyl sulfate as a function of total detergent concentration: 1, detergent alone; 2, detergent with 3 X 10-6 M protein; 3, detergent with 6 x 10-6 M protein. The arrows indicate the apparent c.m.c. in each case. The exact locations of the apparent c.m.c.'s were found by plotting these curves on a large scale. The curves are displaced relative to each other for greater clarity. Each division on the ordinate equals 200 X 10-8 ohm-1 cm.-1. The calculation of r involves small differences
between large numbers, resulting in a low over-all precision. The extent of the experimental errors - is indicated in the figure by the sloping lines passing through each point. The slope of these lines shows additional experiments were made with 6 X lO-5M the correlation existing between r and the concenprotein at detergent concentrations of 2 to 16 X tration of free detergent: since the free detergent lO+M. The value of r was calculated as illus- concentiation is given by d - rp, its calculated trated by the following data from a typical experi- value depends on the value of r. For comparison with our binding results, the ment: p = 3 X lO-5M; d = 8 X M; K = 455 X ohm-' cm.-'; number of faradays data of Pallansch and Briggss are also given in Fig. passed, 8.0 X w 5 ; h N a + = 46.8 Cm.' ohm-l 1. Their results were obtained by equilibrium equiv.-l; AD- = 20.3 cmn2ohm-'equiv.-'; ANa = dialysis at pH 6.8 and ionic strength 0.2. The 0.525 f 0.005 moles per faraday; A P = (2.15 f agreement may be somewhat fortuitous since a t 0.05) X moles per faraday; r = 146 f 10 these low values of r a discrepancy of as much as moles D- per mole protein, by equation 6; concn. 50 per cent. in r would result in only a slight disof free detergent = d - r p = (3.6 f 0.3) X 10-3M. placement of the curves. T o check the binding results obtained by the The results are shown in Fig. 1 where r is plotted against the concentration of free detergent. Each transference method, the number of D- ions bound point represents the average of two or more experi- was determined by a second, independent method ments in which different numbers of faradays were for the special case where the free detergent was used. It was found that ANa and AP were in- at its critical micelle concentration (c.m.c.). dependent of the amount of charge passed if the This method consists of finding the apparent c.m.c. latter was kept small. The results may therefore of the detergent in the presence and absence of be taken as those applying in the limit, when no protein; the shift in c.m.c. corresponds to the charge is passed and where no disturbance of the amount of detergent bound. In each case, the c.m.c. was found by locating the break in the conequilibrium can occur.24 vs. detergent Concentration curve as ductivity (24) With inert electrodes, hydrogen and oxygen is liberated, The described by Williams, Phillips and Mysels for resulting pH changes will alter the charge and thus the mobility of the pure detergent solution^.^^ protein-detergent complex. I n addition all transference numbers will be changed if any H or O H - ions are allowed to migrate from one Experimental conductance curves for sodium compartment t o the other. These difficulties were avoided b y passing M and 6 X dodecyl sulfate with zero, 3 X a small enough charge SO as to keep the pH changes localized around +
the electrodes. Under these conditions the presence of any H + or OH- could be disregarded.
(25) R. J. Williams, J. N. Phillips and K. J. Mysels, Trans. Faradag Soc., 61, 728 (1955).
GEORGESTRAUSS AND ULRICH P. STRAUSS
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Vol. 62
solution so as to lower the detergent/protein ratio, while keeping the protein concentration constant. These new solutions were compared to others of identical composition but which had been made up by adding dry detergent to protein solutions: the specific conductances and intrinsic viscosities of both types of solution were equal. Incomplete reversal would have given conductance and viscosity results measurably different from those actually observed because of the non-linear dependence of these properties on the extent of binding. Non-polar Binding.-To study the affinity of the protein for pure hydrocarbon molecules, the binding of n-heptane and isooctane by serum albumin solutions was measured, using a vapor pressure technique developed in this Laboratory.27 Neither of these hydrocarbons was bound beyond a trace (1-2 moles per mole of protein). This result cannot however be compared directly with the amount of detergent bound since the conditions in the two cases are different: if binding of hydrocarbon a t its saturation vapor pressure is compared with binding of detergent a t its c.m.c., it is evident that a hydrocarbon molecule must be removed from the pure liquid while a detergent molecule must be removed from a micelle. It has been shown28that polysonps such as poly4-vinylpyridine quaternized with n-dodecyl bromide must contain a minimum-sized region of dodecyl groups in order to solubilize aliphatic hydrocarbons. It may therefore be concluded from the present result that serum albumin does not have such large hydrocarbon regions. Degree of Dissociation of the Complex.-The fraction a of Na+ ions dissociated from the complex was calculated by equation 10. The values of a are plotted as a function of r in Fig. 3 (upper diagram). As r increases, the complex acquires an increasingly greater negative charge which should result in a lower degree of dissociation. The results are in agreement with t h i s expectation. Mobility of the Complex.-This quantity, uo,was calculated from K and AP by equation 8. Results are given in the lower diagram of Fig. 3. The mobility of the complex is seen to increase somewhat with increasing r . Putnam and Neurath6 from electrophoresis measurements a t 1O and at ionic strength 0.2 found mobilities of 7.8 and 9.8 X 10-5 volt-1 see-1 a t T = 55 and 110, respectively. The difference between these results and ours is due to the effect of temperature on the viscosity of water (which changes by a factor of 2 between 1 and 25") and to the effect of ionic strength. The relative dependence of mobility on r is similar in both cases. Acknowledgment.-We wish to express our thanks t o the Colgate-Palmolive Company for the financial support of this research, for a post-doctoral fellowship for George Strauss and for providing pure sodium dodecyl sulfate.
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:
34 "
30 0
40
EO
120
160
r. Fig. 3.-The effect of r on the effective degree of digsociation, CY of the protein-detergent complex (upper diagram) and onits mobility, uO,incm.2 volt-' sec.-1, (lower diagram). The size of the circles indicates experimental errors.
M serum albumin are shown in Fig. 2 on a greatly diminished scale. The exact location of the breaks in the curves was found with the aid of large-scale plots which fully utilized the precision of the conductance measurements (better than one part per 1000). The c.m.c. of the detergent in the absence of protein was (8.6 f 0.3) X lod3 M. With 3 X 10-6 M protein, the apparent c.m.c. was (13.3 f 0.4) X M and with 6 X 10-6 M protein it was (17.8 f 0.4) X lod3M.26 From these shifts in apparent c.m.c., r was found to be 157 and 153 for the two protein concentratioiis used. These results are included in Fig. 1. They agree, within the experimental error, with the transference results and thus confirm the latter. The results from both these methods show that the equilibrium between bound and free detergent is independent of the protein concentration at least up to 6 X molar. Reversibi1ity.-Complexing between protein and detergent was found to be completely reversible a t r values up to 160. Reversibility was demonstrated by making up proteiii-detergent solutions and then diluting them with additional protein (2G) For pure detergent solutions, the slope of the conductivity curve is constant below the c.1n.c. and changes to a smaller constant value over a small concentration range a t the c.m.c. With proteindetergent solutions, the change in slope occurs over a considerable concentration range. In order t o treat the d a t a consistently, and since shifts in c.m.c. rather than absolute values were of intcrest, the c.m.c. in each case wa8 taken as the point at which the slope became constant. With pure detergent, this procedure gave a c.1n.c. somewhat higher than t h a t obtained by extrapolating the straight portiona of the conductivity curve until they intersected. The latter procedure gave a c.m.c. of 8.25 X 10-8 M. The value previously reported26 is 8.2 x 10-3 M .
(27) H. L. Dragun, private communication. (28) U. P. Strauss and N. L. Gershfeld, THISJOURNAL, 68, 747 (1954).
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