Effects of Surface Charge Distribution of Proteins in Their

Langmuir 2000, 16, 3133-3140

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Effects of Surface Charge Distribution of Proteins in Their Complexation with Polyelectrolytes in an Aqueous Salt-Free System Daisuke Takahashi,† Yuichi Kubota,† Kenji Kokai,† Tsuyoshi Izumi,† Mitsuo Hirata,† and Etsuo Kokufuta*,‡ Department of Industrial Chemistry, College of Industrial Technology, Nihon University, Narashino, Chiba 275-8575, Japan, and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan Received August 17, 1999. In Final Form: January 3, 2000 Complexation between protein and polyelectrolyte was studied by a combination of three main experimental techniques: turbidimetric titration, quasi-elastic light scattering (QELS), and static light scattering (SLS). Proteins of two sorts, lysozyme (Lyz) and ribonuclease (RNase), were chosen by considering the following characteristics: (i) Both proteins have the same number (19) of basic groups; (ii) their distribution is almost homogeneous on Lyz but not on RNase; (iii) there is little difference in the molar mass between both proteins. Potassium poly(vinyl alcohol) sulfate (KPVS) with different molecular weights (M h PE) and various degrees of esterification (De) was used as the polyelectrolyte. We employed a salt-free aqueous medium and adjusted it to pH 2, the level of which forces to completion the protonation of all of the basic groups. As the titration of proteins with KPVS proceeded, the absorbance (A) as an indication of turbidity increased linearly and then rapidly at a certain titrant volume, referring to the end point of titration. The slope of the linear plots of A vs titrant volume for Lyz was little dependent on M h PE and De. In the case of RNase, however, the slope increased with decreasing De and with increasing M h PE. From the studies of the hydrodynamic radius by QELS as well as the molecular weight and radius gyration by SLS, it was found that the M h PE and De effects observed in the titration curve correspond to the changes of both size and mass of aggregated “intrapolymer” complexes formed during titration. Thus, we estimated the degree of aggregation (R) through dividing the mass from SLS by the calculated mass of an intrapolymer complex. This showed that R decreases with increasing De in both protein systems, while increasing M h PE decreases R in the Lyz system but does not change it in the RNase system. These results were discussed in detail by considering the polarizability of the intrapolymer complex, the level of which is affected by the complementarity of the spacing between charges on the protein to the uniform spacing between polymer charges.

Introduction When adding polyelectrolytes to an aqueous protein solution in the presence or the absence of salts such as NaCl, the proteins bind to a polyion via Coulomb attraction, yielding protein-polyelectrolyte complexes (PPCs).1,2 Of particular interest would be the way in which globular proteins bind to a flexible polymer chain, an understanding of which could provide a better explanation of the PPC formation mechanism. Our previous studies1,3-9 on PPC formation have employed a salt-free system at a pH level * To whom correspondence should be addressed. † Nihon University. ‡ University of Tsukuba. (1) Kokufuta, E. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Davis, R., Thies, C., Bock, J., Schulz, D., Eds.; Springer-Verlag: Heidelberg, 1993; Chapter 18. (2) Xia, J.; Dubin, P. L.; Ahmed, L. S.; Kokufuta, E. In Macro-ion Characterization: From Dilute Solutions to Complex Fluids; Schmitz, K. S., Ed.; American Chemical Society: Washington, DC, 1994; Chapter 17. (3) Kokufuta, E.; Shimizu, H.; Nakamura, I. Polym. Bull. 1980, 2, 157. (4) Kokufuta, E.; Shimizu, H.; Nakamura, I. Macromolecules 1980, 14, 1178. (5) Kokufuta, E.; Shimizu, H.; Nakamura, I. Macromolecules 1981, 15, 1618. (6) Kokufuta, E., Takahashi, K. Polymer 1990, 31, 1177. (7) Izumi, T.; Hirata, M.; Takahashi, K.; Kokufuta, E. J. Macromol. Sci., Pure Appl. Chem. 1994, A31, 39. (8) Kokufuta, E. Prog. Polym. Sci. 1992, 17, 647 and references therein. (9) Tsuboi, A.; Izumi, T.; Hirata, M.; Xia, J.; Dubin, P. L.; Kokufuta, E. Langmuir 1996, 12, 6295.

which forces to completion either the protonation of basic groups or the deprotonation of acidic groups bound to a protein. Under such experimental conditions, the formal charge of a protein should be equivalent to the number of acidic or basic groups per protein; therefore, we may calculate from the amino acid composition the number of ionic groups taking part in the complexation. Then it was possible to demonstrate that a number of proteins form PPCs with polyions through a 1:1 stoichiometric binding between oppositely charged groups.1,3-9 The resulting PPCs were insoluble in water, and thereby the system underwent a phase separation during the course of the complexation. However, there was an appreciable retention of biochemical function in the resultant PPCs;6,7 thus, it has been suggested that such a PPC has great potentialities as an immobilized enzyme.1,8 On the basis of the above experimental results, we have discussed the formation mechanism of PPCs.9 It was suggested that, upon addition of polyions, the binding of proteins to a polyion results in an “intrapolymer” complex,10 in which the polyion charges are balanced by the stoichiometric neutralization with the opposite charges (10) Prior to our report (ref 9) on the formation of an intrapolymer PPC in a salt-free system, it was known that intrapolymer PPCs are formed in a salt-containing system (Xia, J.; Dubin, P. L.; Dautzenberg, H. Langmuir 1993, 9, 2015). On the other hand, our recent study has demonstrated that the complexation of protein with a “neutral” polymer such as poly(ethylene glycol) gives forth a very stable and water-soluble intrapolymer complex (Azegami, S.; Tsuboi, A.; Izumi, T.; Hirata, M.; Dubin, P. L.; Wang, B.; Kokufuta, E. Langmuir 1999, 15, 940).

10.1021/la991108z CCC: $19.00 © 2000 American Chemical Society Published on Web 02/18/2000

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of proteins. Such intrapolymer PPCs subsequently associate with one another to yield neutral aggregates (AG1). Although attempts to isolate the intrapolymer PPCs have not been successful as yet, we may characterize AG1 by means of turbidimetric titration, quasi-elastic light scattering (QELS), static light scattering (SLS), and electrophoretic light scattering (ELS). In particular, a combination of the titration, QELS, and SLS has demonstrated that addition of a very slight amount of polyions to a protein solution with a fixed concentration brings about a linear increase in the molecular weight (M h x), hydrodynamic radius (Rh), and radius gyration (Rg) of AG1. Further addition of polyions causes an increase in the concentration of AG1 without change in its particle size. When the concentration of AG1 exceeds a certain level, larger aggregates (AG2) form through association of AG1 (see Figure 12 in ref 9). Therefore, it would be possible to see how the charge distribution of proteins and the linear charge density of polyions affect the formation processes as well as the structure of AG1. Through this, one may discuss the formation mechanism of intrapolymer PPCs at the molecular level. Proteins of two sorts, lysozyme (Lyz) and ribonuclease (RNase), were chosen in this study because the following features are known (see ref 11): (i) Both proteins have the same number (19) of basic groups; (ii) their distribution is almost homogeneous on Lyz but not on RNase; (iii) there is little difference in the molecular weight between both proteins. Also chosen in this study was potassium poly(vinyl alcohol) sulfate (KPVS) with different chain lengths and with various levels of linear charge density. Dubin et al.12 have studied effects of protein charge heterogeneity and polymer charge density in PPC formation in saltcontaining solutions with different pHs using bovine serum albumin, Lyz and RNase. They used polyelectrolytes of different sorts to vary the linear charge density, while we employed polymers of the same sort whose linear charge densities are exactly controllable by a combination of the following two reactions: the introduction of ionizable -OS(dO)2OH groups into poly(vinyl alcohol) (PVA) via its esterification with sulfuric acid and the removal of a part of the introduced ionizable groups in KPVS via its hydrolysis with HCl. Experimental Section Proteins. Lyz from chicken egg white and RNase from bovine pancreas were obtained from Sigma Chemical Co. as 95-99% pure lyophilized proteins. Polyelectrolytes. KPVS samples of various linear charge densities were prepared via the esterification of three PVAs which were commercial products (Kurare Co., Ltd., Tokyo, Japan) with the degree of polymerization (DP) of 500, 1500, and 3500. The esterification of PVA was performed in a chlorosulfuric acidpyridine mixture at 70 °C (see R1-R3).13 Pyridine (60 mL) was placed in a 500 mL Pyrex Erlenmeyer flask and cooled in an ice bath. After that, chlorosulfuric acid (16 mL) was added dropwise to the flask and vigorously stirred to form pyridinium salt (I; see R1). Finally, 10 g of PVA was dissolved with stirring to react (11) We obtained information about both proteins from the Protein Data Bank, Oct 1996, Release No. 78, Brookhaven National Laboratory, Upton, NY. For details of these data bank, see: Abola, E. E.; Manning, N. O.; Prilusky, J.; Stampf, D. R.; Sussman, J. L. J. Res. Natl. Inst. Stand. Technol. 1996, 101, 231. (12) (a) Park, J. M., Muhoberac, B. B., Dubin, P. L., Xia, J. Macromolecules 1992, 25, 290. (b) Mattison, K. W.; Dubin, P. L.; Brittain, I. J. Phys. Chem. B 1998, 102, 3830. (13) Takahashi, A., Nagasawa, M., Kagawa, T. Kougyo Kagaku Zasshi (an article published by Chemical Society of Japan) 1958, 61, 1614 (in Japanese).

Takahashi et al. with I (see R2). The reaction was continued at 70 °C until a desired degree of esterification was obtained. The resulting reaction mixture was treated with an aqueous KOH solution (1 M) to release pyridinium chloride (see R3) and then poured slowly into a large volume of methanol to precipitate the polymer, which was separated by filtration, purified by reprecipitating two times from water with methanol, and dried under vacuum.

For the polymer with a low degree of esterification, its charge density was accurately regulated via partial hydrolysis of KPVS with HCl (see R4). An aqueous solution (10 mL) containing desired

amounts of KPVS and HCl was prepared using pure water as the solvent and sealed in a glass ampule under vacuum. The reaction was continued at 50 °C until the esterification degree came up to a desired level. After completion of the reaction, the polymer was precipitated with methanol, dissolved again in pure water, and dialyzed against a large volume of pure water. The dialyzed solution was lyophilized and dried at 50 °C under vacuum. Complexation. The formation of PPCs was examined by means of turbidimetric titration. Salt-free aqueous sample solutions (30 mL) with a fixed protein concentration were titrated at 25 °C in a cubic 2.6 cm path-length glass cell with a KPVS solution as the titrant. Unless otherwise noted, the concentrations of protein and KPVS were 0.1 and 0.415 mg/mL, respectively. To avoid changes in pH during titration, both protein and polyelectrolyte solutions were adjusted to the same pH level. The samples for QELS, SLS, and ELS were obtained at different stages of the titration; that is, the protein solution into which different amounts of KPVS had been added was used for these light scattering experiments. Details of experimental techniques have been reported in ref 9. Measurements. All of the measurements were performed at 25 °C in the same way as reported in ref 9: turbidimetric titration with a Hirama automatic recording titrator (model ART-3); QELS and SLS with a Brookhaven system (Holtsville, NY) equipped with a 256-channel digital autocorrelator (BI-2030 AT) and a 2 W Ar+ ion laser (Stabilite 2017, Spectra-Physics Lasers); ELS with a Coulter DELSA 400 apparatus (Hialeah, FL); refractive index measurements with an Otsuka electrophotometric differential refractometer (model DRM-1021). We also employed the colloid titration for determining the esterification degree of

Effects of Surface Charge Distribution of Proteins

Langmuir, Vol. 16, No. 7, 2000 3135

Table 1. Properties of KPVS Samples De

M h PE (104 g mol-1)

Rg (nm)

Rh (nm)

F ()Rg/Rh)

A2 (10-4 cm3 mol g-2)

dn˜ /dc (mL g-1)

KPVS (5/78)

0.778

6.1

21

12

1.75

4.04

0.1073

KPVS (15/13) KPVS (15/33) KPVS (15/53) KPVS (15/76) KPVS (15/92)a

0.130 0.328 0.530 0.760 0.922

8.9 12.1 14.9 18.1 18.7

22 27 31 33 35

15 18 20 21 22

1.47 1.50 1.55 1.57 1.59

4.12 2.18 3.51 3.71 7.24

0.1314 0.1175 0.1095 0.0988 0.0937

KPVS (35/76)

0.765

46.6

60

36

1.67

7.85

0.0977

sample

a

Measured for a newly prepared sample, so that there are some differences between the data reported here and in ref 9.

KPVS. The standard titrant used was an aqueous solution containing 4 mM (based on the cationic groups) of 6-O-[(2hydroxyethyl)-2-(trimethylammonio)chitosan iodide, which was commercially obtained from Wako Pure Chemical Co.

of A against titrant volume (Vt in mL) may be expressed as

A)H Results Characteristics of KPVS. We prepared seven samples of KPVS whose charge densities as well as chain lengths are different from one another. These polyelectrolytes were then characterized very carefully by studying the following quantities: molecular weight (M h PE), Rg, and the second virial coefficient (A2) by SLS; Rh by QELS; and the degree of esterification (De) by colloid titration. The results were shown in Table 1, together with the change in refractive index with concentration, (dn˜ /dc)PE, the value of which is required for the analyses of SLS data for both the polymer and the complex. In our SLS measurements, the angular dependence of scattered light was studied over a range of polyelectrolyte concentration (CPE) from 0.075 to 4.0 mg‚mL-1. Then, the Zimm plots gave two straight lines with correlation coefficients > 0.99, co-intersecting the Y axis, enabling us to determine M h PE and Rg. The logarithmic plots of Rg as well as Rh against M h PE showed a good linear relation (correlation coefficients > 0.98); that is, log Rg ) 0.53 log h PE - 1.42 (see ref 14). M h PE -1.27 and log Rh ) 0.52 log M The values of DP for PVA are known, and a relation among DP, De, and M h PE can be given by M h PE ) DP{M1De + M2(1 - De)}, where M1 ()162) and M2 ()44) are respectively the molecular weights of -CH2CH(OSO3K)and -CH2CH(OH)- residues. Then we may obtain M h PE ) 1.77 × 105De + 6.6 × 104 for a series of KPVS (15/13) to KPVS (15/92). The M h PE calculated by this relation was found to well agree with that determined by SLS at De < 0.5. At De > 0.5, however, the values by SLS are 80-90% of the calculation, for example, 90% for KPVS (15/76) and 82% for KPVS (15/92). This is caused by the depolymerization which would take place to some degree during the esterification, because we were forced to continue the reaction over several hours to obtain a high De value. Turbidimetric Titration Curves. We have theoretically dealt with the turbidimetric titration curve for the PPC formation process;9 that is, there is a change in the absorbance (A) because of the appearance of turbidity15 while a protein solution is titrated with a polyelectrolyte solution. Assuming that the PPC formation takes place via a 1:1 stoichiometric binding between the opposite charges attached to the protein and the polyion, the plots (14) The logarithmic plots of Rg against molecular weight for PVA in pure water gave the slope of 0.56 (see: Wang, B.; Mukataka, S.; Kodama, M.; Kokufuta, E. Langmuir 1997, 13, 6108). Taking this into account, we may say that the polyion charges are fully eliminated in 0.2 M NaCl used as the solvent for our SLS and QELS measurements. (15) A relation between turbidity (τ) and absorbance (A) is given by τ ) 2.3A/l, where l is cell length.

( )( ){ (

) }

n j 2Mpro l CPE M h PE + 2n j+ Mpro Vt 2.3 Vi M h PE

(1)

j is the where Vi denotes the volume of protein solution, n average number of bound protein molecules per one polyion chain, and Mpro is the absolute molecular weight of the proteins. The proportionality constant (H) is then written as

H ) (32π3/3λ04NA)n˜ 02(dn˜ /dc)x2

(2)

Here, λ0 is the wavelength of light in a vacuum, NA is Avogadro’s number, n˜ 0 is the refractive index of the medium, and (dn˜ /dc)x is a change in the refractive index with PPC concentration (Cx). One may predict from eq 1 that the titration of proteins with polyions would cause j remains unchanged a linear increase in A with Vt, when n during the titration. A linear relationship between A and Vt has been found in our titration with KPVS (15/92) of various proteins in a salt-free system at pH 2, at which all of the protein-bound basic groups are fully protonated.9 Then we also found that A abruptly increases when Vt reaches a certain volume (V′t), which can be viewed as the Vt at the end point of the titration because (i) V′t is a linear function of protein concentration (Cpro) and (ii) the relation between V′t and Cpro is given by

V′t ) NcCproVi(M°/103CPE)

(3)

where Nc (in equiv/g) denotes the number of cationic charges per unit mass of protein and M° is the molecular weight per ionizable group (i.e., equivalent weight in g/equiv) for KPVS. As a result, our titration in ref 9 strongly suggested that an electrically neutral PPC with a constant size (i.e., AG1 mentioned in the Introduction) is formed by addition of KPVS to protein solutions (this was supported by a combination of QELS and ELS; see ref 9). Taking the above into account, we studied the process of PPC formation by means of turbidimetric titration. Figures 1 and 2 show the titration curves with KPVS h PE for Lyz and RNase, respechaving different De and M tively. A linearity was observed in the A vs Vt plots for all of the titration curves. In the Lyz system, the slope was little dependent on the polyion charge density (Figure 1a) as well as on the chain length (Figure 1b). In the case of RNase, however, the slope increased with a decrease in the polyion charge density (Figure 2a) as well as with an increase in the chain length (Figure 2b). Thus, it would be interesting to compare each value of the slopes in Figures 1 and 2 with the calculation from eq 1. Then (dn˜ / dc)x in eq 2 was obtained using a relation of (dn˜ /dc)x ) {1/(1 + β)}(dn˜ /dc)PE + {β/(1 + β)}(dn˜ /dc)pro, where (dn˜ / dc)PE and (dn˜ /dc)pro are changes in the refractive index

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Figure 1. Turbidimetric titration curves of Lyz with KPVS having different De (a) and different M h PE (b). Vi ) 30 mL; Cpro ) 0.1 mg/mL; CPE ) 2.5 × 10-3 mol/L (based on the ionizable groups). By substituting these values for eq 3, we obtain V′t ) 1.63 mL.

with concentration for polyelectrolyte and protein, reh PE (i.e., the mass ratio of bound spectively, and β ) n j Mpro/M protein to polymer). Because all of the other parameters in eqs 1 and 2 are known, we may obtain the theoretical slope for each titration curve, for example, 1.64 × 10-4 mL-1 for Lyz with KPVS (15/13) and 1.51 × 10-4 mL-1 for RNase with KPVS (15/13). The results obtained were 2 orders of magnitude smaller than those in Figures 1 and 2. The reason for this disagreement is, however, understandable considering that the calculation is made for the

Takahashi et al.

Figure 2. Turbidimetric titration curves of RNase with KPVS having different De (a) and different M h PE (b). All of the titration conditions are the same as those used for Lyz, so that V′t ) 1.63 mL.

corresponding intrapolymer complexes. In other words, the slopes in Figures 1 and 2 would be assigned to an aggregate (i.e., AG1) of intrapolymer complexes. Indeed, the experimentally obtained slope is consistent with the product of the calculation and the aggregation degree (R; see Table 2 in the next section), for example, 0.046 mL-1 for Lyz with KPVS (15/13) and 0.022 mL-1 for RNase with KPVS (15/13). Thus, the following are suggestible: (i) The aggregates of intrapolymer PPCs are formed during the titration, and (ii) the aggregation process is influenced by the surface charge distribution of proteins as well as by


Langmuir, Vol. 16, No. 7, 2000 3137

systems. Because the weight-average molecular weight (M h °x) of an intrapolymer PPC may simply be written as

M h °x ) M h PE + n j Mpro

(4)

we obtain the degree of aggregation (R) from

h °x R)M h x/M

Figure 3. Change in electrophoretic mobility (U) during the course of the titration for RNase with KPVS (35/76). The titration curve has been shown in Figure 2b. The full line denotes the mobility for the titrated samples which were obtained at the different stages of the titration. The dashed line shows the mobility for the samples after removal of most of PPCs by passing the titrated sample through a 0.2 µm filter. At Vt ) 1.3 mL, it was possible to detect the free protein (peak c; U ∼ 0.92 µm cm V-1 s-1) in the filtered sample (see the dashed line) but not in the titrated sample (see the full line) because of a strong light scattering intensity of PPC particles (peak b; U ∼ 0 µm cm V-1 s-1). At Vt ) 1.63 mL corresponding to V′t, however, there was no free protein in either the filtered and the titrated sample, therefore, indicating that all of the proteins were consumed for forming PPC at the end point of the titration. At Vt ) 2.0 mL (>V′t), excess KPVS (peak a; U ∼ 2.8 µm cm V-1 s-1) was detected in the filtered sample.

both the linear charge density and the chain length of polyelectrolytes. In our titration, both CPE and Cpro were exactly adjusted, so as to become V′t ) 1.63 mL. Thus, all of the titration curves for Lyz exhibit a rapid increase in A at V′t ∼ 1.63 mL. In the case of RNase, however, the onset of such a rapid increase in A is shifted to a low Vt range when increasing M h PE or decreasing De (see full titration curves in parts a and b of Figure 2). In addition, turbidity around V′t becomes stronger in the RNase system than in the Lyz system. From these, one may argue that eq 3 fails for the complexation of RNase with a polyelectrolyte having a low charge density or a high molecular weight. In other words, we may not assume the electroneutrality in the complexes of RNase with such a polymer as KPVS (35/ 76). Nevertheless, as shown in Figure 3, our ELS experiments for the RNase-KPVS (35/76) system clearly demonstrated that (i) at Vt < V′t there is no particle other than the neutral PPC and the uncomplexed free protein with positive charges and (ii) at Vt ) V′t there is no “charged” particle. Therefore, the characteristics observed in the full titration curves for RNase may be related to a difference in the aggregation processes of intrapolymer PPCs arising from Lyz and RNase (see the Discussion section). QELS and SLS. It has been suggested that PPCs formed during the titration are the aggregates of intrapolymer complexes, referring to AG1. We thus employed QELS and SLS to examine the molecular characteristics for such PPCs. Our preliminary experiments with QELS showed that the size of PPC remains constant at different stages of the titration at Vt < V′t; therefore, the SLS study was performed at Vt ) 0.25 V′t for all of the titration

(5)

The results of QELS and SLS are summarized in Table 2. As expected, there are marked differences between the Lyz and RNase systems. The following are the important features that should be considered in our discussion about the PPC formation mechanism: (i) In the Lyz system, the mass (M h x) as well as the size (Rh and Rg) of the complexes h PE, while increases in these is little varied with De and M factors reduce the aggregation degree. (ii) Both the size and the mass, as well as the aggregation degree, of the RNase complexes are reduced with increasing De. (iii) In the RNase system, an increase in M h PE increases both size and mass, but little changes the aggregation degree. (iv) The values of F (i.e., Rg/Rh) are larger in the RNase system than in the Lyz system. Discussion General Aspects for PPC Formation in the Lyz and RNase Systems. Before discussion of the above results in connection with the PPC formation mechanism is opened, it would be convenient to make clear a difference in the surface charge distributions between Lyz and RNase. For this purpose, we used a protein molecular model and turned it around a fixed axis through a full 360°. Then we can see all of 19 basic groups bound to Lyz and to RNase (see Figure 4). Moreover, these basic groups are found to be distributed almost randomly on the Lyz but locally on the RNase, although both proteins have the same number of basic groups as well as almost the same molecular weight (Mpro ) 14 306 for Lyz and 13 682 for RNase). Now let us consider how the protein charge distribution affects the formation of PPCs with polyions having different linear charge densities and different molecular weights. As mentioned in the Introduction, there are three stages in the PPC formation: formation of intrapolymer PPC, its aggregation to form AG1, and further aggregation of AG1 to yield AG2. Regarding the intrapolymer PPC formation, we should take into consideration that the spacing between charges on the protein is not variable as well as not enough to be complementary to the uniform spacing between the polyion charges. For this reason and also from considerations of restrictions on polymer chain configurational entropy, we cannot imagine tight ion pairing in the intrapolymer PPC. Rather, we have to consider the proteins in the PPC as “loosely” bound and the ion pairs as labile and prone to reconfiguration. Furthermore, there must be local regions of excess positive and negative charges within the PPC. Thus, while the intrapolymer PPC may have net electroneutrality, it is highly “polarizable”. This polarizability seems to promote the aggregation of intrapolymer complexes to form AG1. The amount of polarization for the intrapolymer PPC from Lyz should be larger than that from RNase, because the complementarity of the spacing between charges on the protein to the uniform spacing between polymer

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Takahashi et al.

Table 2. Molecular Characteristics of Lysozyme and Ribonuclease Complexes with KPVS run no.

protein

KPVS

M h x (107 g mol-1)

M h °x a (105 g mol-1)

Rb

Rg (nm)

Rh (nm) (nm)

F ()Rg/Rh)

1 2 3 4 5

Lyz Lyz Lyz Lyz Lyz

KPVS (15/13) KPVS (15/33) KPVS (15/53) KPVS (15/76) KPVS (15/92)

6.69 7.14 7.29 7.33 7.41c

2.36 4.82 7.06 9.54 10.4

284 148 103 77 72c

66 71 75 77 79c

50 51 52 52 52c

1.32 1.39 1.44 1.48 1.52

6 7

Lyz Lyz

KPVS (5/78) KPVS (35/76)

7.46 7.21

3.24 24.6

230 29

75 69

53 50

1.42 1.38

8 9 10 11 12

RNase RNase RNase RNase RNase

KPVS (15/13) KPVS (15/33) KPVS (15/53) KPVS (15/76) KPVS (15/92)

3.40 3.20 2.70 1.90 0.664c

2.29 4.66 6.82 9.21 9.99

148 69 40 21 7c

84 76 71 65 51d

50 47 44 41 34c

1.68 1.62 1.61 1.59 1.50

13 14

RNase RNase

KPVS (5/78) KPVS (35/76)

0.611 6.14

3.12 23.8

20 26

57 92

37 50

1.54 1.84

a Calculated by eq 4. b Calculated by eq 5. c There is a little difference between the data reported here and those in ref 9 because KPVS (15/92) was newly prepared. d Showed the mean from five measurements with different samples, because the experimental uncertainty (∼13%) for the KPVS (15/92)-RNase system was larger than those (less than 7%) for the other systems (this seems to be due to an extended structure of the intrapolymer complex which is constituted of a large number of RNase molecules bound to a KPVS ion).

charges is poorer in the former than in the latter (see ref 16). This is quite obvious from the models of Lyz and RNase in Figure 4. As a result, the intrapolymer PPC from Lyz would have a greater tendency to aggregate one another, compared with that from RNase. h PE, it is predictable that Regarding effects of De and M the polarizability of intrapolymer PPCs from the same protein is changed by De but not by M h PE. The reason is that the spacing between polymer charges is inversely proportional to the linear charge density, whereas the chain length does not vary the spacing. Therefore, De may cause a change in the aggregation of the intrapolymer PPC, the magnitude of which should be larger in the Lyz h PE system than in the RNase system. In contrast to De, M has little or no influence on the aggregation when De remains constant. Effects of De. Our experiments have demonstrated that the size of PPC remained unchanged over a wide Vt range where the titration curve exhibited a linear increase. Under such a condition, the mass of all of the PPCs was larger than that of the corresponding intrapolymer PPCs; h °x and hence R > 1. This means that the that is, M hx>M PPC formed during the titration can be assigned to AG1 with a definite size. In the RNase system, a decrease in the size of AG1 with increasing the polyion charge density is found from Figure 2a as well as from runs 8-12 in Table 2. Another important finding in the RNase system is that an increase in the polyion charge density strongly inhibits the aggregation of intrapolymer PPC. Such an inhibition is also found in the Lyz system, but the values of R for Lyz are considerably larger than those for RNase (see runs 1-5 in Table 2). In addition, in the Lyz system there is little change in the size of AG1. These differences clearly mean that the amount of polarization for the intrapolymer PPC resulting from RNase is smaller than that from Lyz; this is just as we have expected in the above section. (16) A mean distance between anions bound to KPVS is roughly estimated to be 11.5 Å at De ) 0.13 and 1.6 Å at De ) 0.92 from the length of the C-C bond (1.5 Å). On the other hand, a hydrodynamic diameter is about 40 Å for both proteins (see Table 2 of ref 9). Thus, from Figure 4 it is found that several charges on Lyz are located at an unfavorable spacing which is not complementary to the spacing between the polymer-bound anions, for example, the spacing between positions 73 and 97 as well as between positions 73 and 112. In contrast, most of the charges on RNase are located at favorable positions at which some chance of forming ion pairs with a polyanion is expectable, with a few exceptions (e.g., the spacing between positions 39 and 91).

The charge density of KPVS varies n j in eq 4 because De leads to the change of n j as given by

n j)

( )(

)

M h PEDe 1 Zpro DeM1 + (1 - De)M2

(6)

where Zpro ()19) is the number of charges per protein, and the term in the second set of parentheses is equivalent to the formal charges of KPVS. A combination of eqs 4 and 6 shows that M h °x should be increased with increasing De, while the experiments indicate that an increase in De decreases R. Thus, it is predictable that when the increase in M h °x and the decrease in R compensate for each other, the mass as well as the size of AG1 is little or not influenced by De. This is the case in the Lyz system but not in the RNase system. Thus, we may consider that (i) there is a thermodynamically stable size of AG1 with a definite mass and (ii) such a size would be attained by dissociation of a part of the bound proteins followed by their reassociation within the AG1 to minimize the amount of polarization during the aggregation of intrapolymer PPCs.17 Such dissociation and reassociation should occur more easily in the Lyz system than in the RNase system; thus, the former exhibited little change in the size as well as in the mass with the polyion charge density. As a result, it is reasonable to consider that AG1 in the Lyz system prefers a random-coil structure rather than an extended structure. This can be supported by the fact that the change in F with De for Lyz is within a range of 1.3-1.5 (see Table 2 and ref 18). We must explain why an electrically neutral AG1 aggregates to form AG2 before reaching V′t, when RNase was titrated with the polymers, other than KPVS (15/92) (see Figure 2). The AG1 in the RNase system prefers an extended structure (F > 1.5; see Table 2). As the titration proceeds to increase the concentration of such an extended AG1, its aggregation due to hydrophobic interaction and/ or hydrogen bonding would occur easily, yielding AG2 with a very large particle size (this is the reason for the (17) The size (Rh) of AG1 remained unchanged when the sample obtained from the titration (Vt ∼ 1.0 mL) of Lyz or RNase with KPVS (5/78) was diluted five times with a 0.01 M HCl solution (pH 2) as well as when the diluted sample solution was allowed to stand for 1 week. These indicate a high stability of AG1. (18) Hydrodynamic theory (Konishi, T.; Yoshizaki, T.; Yamakawa, H. Macromolecules 1991, 24, 5614) shows that F changes from infinity to 0.775 when the polymer structure changes from a long rod to a sphere, with values from 1.3 to 1.5 for random coils.


Langmuir, Vol. 16, No. 7, 2000 3139

Figure 5. Changes in the degree of aggregation (R), radius of gyration (Rg), and hydrodynamic radius (Rh) for PPCs formed at the early stages of the titration with KPVS (5/78) of Lyz (open circle) and RNase (closed circle). Error bars show the range between maximum and minimum values from five measurements with different samples. The titrant volume was normalized by V′t.

Figure 4. Distribution of 19 basic groups on the surfaces of Lyz and RNase. The numerals denote the location of basic amino acid residues appearing in the diagrams regarding the amino acid sequence of Lyz or RNase (see ref 11). The N-terminal amino acid for both proteins is the lysyl residue; thus, the R and amino groups are indicated by the same numeral (i.e., 1).

appearance of a strong turbidity in the titration curves of RNase). As a result, we observed the onset of an abrupt increase in A at Vt < V′t but not around V′t. Effects of M h PE. In the RNase system, an increase in M h PE under a fixed De (∼0.77) brought about little change in R (Table 2, see runs 13, 11, and 14 in order); this aspect is just as we had expected. In addition, a linear increase in both the size and mass of AG1 is explicable in terms of the M h PE-dependent change in M h x° (see eq 4). In the case

of Lyz, however, M h PE led to a few changes in both mass and size but to a marked change in R (runs 6, 4, and then 7 in Table 2). Thus, we may say again that in the Lyz system in which the resulting intrapolymer PPC is highly polarizable, the increase in n j and the decrease in R compensate for each other, yielding AG1 with a thermodynamically stable size. Effect of Protein Charge Distribution on Formation of Intrapolymer PPC. The formation of an intrapolymer PPC should be expected if we add one polyion into an aqueous protein solution; however, such a way is impossible in experiments. In addition, many difficulties would arise in the isolation of intrapolymer PPCs from their mixtures with free proteins. For these reasons, we cannot make use of intrapolymer PPCs from Lyz and RNase to study their molecular properties. Nevertheless, our previous study9 has demonstrated that the aggregation process of an intrapolymer PPC can be observed by QELS and SLS, when very slight amounts of KPVS (15/92) were added into an aqueous solution of human serum albumin (HSA) having 100 basic groups on its surface. Taking the above into account, we attempted to study the aggregation process of intrapolymer PPCs at the beginnings of the titration for Lyz and RNase using KPVS (5/78) as the titrant. Both QELS and SLS were available for this purpose, although it was difficult to perform an

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Takahashi et al.

Table 3. Molecular Characteristics for Lysozyme and Ribonuclease Complexes Obtained with Infinitesimal an Quantity of KPVS protein

R′

M h ′x (105 g mol-1)

R′g (nm)

R′h (nm)

F′

Lyz RNase

15 1

49.1 3.15

39 22

27 11

1.44 2.00

accurate measurement of turbidity (A < 0.004). As can be seen from Figure 5, the values of R, Rh, and Rg linearly increase against the normalized titrant volume (Vt/V′t). This allows us to extrapolate each line to Vt/V′t f 0 and to study the molecular characteristics for a PPC arising from an infinitesimal quantity of KPVS. The values obtained by extrapolation were then distinguished by putting a dash from those in Table 2 and summarized in Table 3. It is found that in the RNase system R′ ∼ 1, i.e., h °x (3.12 × 105), suggesting the formation of an M h ′x ∼ M intrapolymer PPC. Another important finding of RNase is that the value of R′g as well as R′h is very close to that for the uncomplexed KPVS (5/78). Thus, it appears that the binding of RNase molecules to one KPVS ion little accompanies a conformational change of the polyion, because any contraction due to local collapse of polymer segments on the protein surface is compensated for by interprotein steric repulsion. This is just the same aspect as that observed in the complexation between HAS and KPVS (see ref 9). In contrast to RNase and HSA, M h x′ for Lyz is 15 times M h °x (3.24 × 105; for the intrapolymer complex) and 1/15 M h x (7.46 × 107; for AG1). Moreover, F′ for Lyz is not different from F for AG1. These clearly show that the intrapolymer PPC arising from Lyz immediately aggregates due to its high polarizability. From the results in this study and also in the previous study9 for HSA, it is very clear that the complementarity of the spacing between charges on the protein to the uniform spacing between polymer charges should be a primary factor for determining the stability of an intrapolymer PPC. HSA has 100 positive charges, while only 19 positive charges bind to RNase. Nevertheless, the spacing between charges on RNase is not so different from that on HSA, because the basic groups on RNase are concentrated within a narrow area of its surface. Thus, not only HSA but also RNase results in a stable in-

trapolymer complex with an infinitesimal quantity of KPVS, at least De > 0.7. In contrast to these proteins, Lyz has the basic groups which are scattered over its surface. For this reason, even if an intrapolymer complex results in the Lyz system, it is highly polarizable and immediately aggregates. Conclusions Effects of surface charge distribution of proteins in their complexation with polyions having a difference in the linear charge density and in the chain length were studied by a combination of turbidimetric titration and light scattering techniques. For this purpose, Lyz and RNase were used because both proteins have the same number (19) of basic groups and little difference in the molar mass but a clear difference in the charge distribution. From the titration of aqueous salt-free protein solutions (pH 2) with h PE a variety of KPVS titrants having different De and M values, it was found that all of the resultant PPCs are aggregates (AG1) of the corresponding intrapolymer PPCs. When effects of M h PE and De on both size and mass as well as on the aggregation of AG1 were studied in detail, the following features became apparent: (i) Both size and mass of AG1 from the Lyz complex were little influenced by M h PE and De, while the aggregation degree was reduced h PE under the condition where when increasing De and M one of both factors was fixed. (ii) In the RNase system, an increase in De brought about a decrease in both size and mass as well as in the aggregation degree. (iii) Both size and mass of AG1 from the RNase complex increased in h PE did not affect the aggregation. proportion to M h PE, but M These results are well explained in terms of the polarizability for the resulting intrapolymer PPCs, the level of which is affected by the complementarity of the spacing between charges on the protein to the uniform spacing between polymer charges. As a result, the present study could provide a key for understanding the mechanism of PPC formation at the molecular level. Acknowledgment. This research was supported by a grant to E.K. from the Ministry of Education of Japan (Grant 08455434). LA991108Z

Effects of Surface Charge Distribution of Proteins in Their

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