538
Bioconjugate Chem. 1999, 10, 538−543
TECHNICAL NOTES Direct Observation of Fibrinogen-Heparinoid Complexes Formation Using Surface Plasmon Resonance Nobuyuki Sakamoto, Tomoko Shioya, Takeshi Serizawa, and Mitsuru Akashi* Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan. Received December 26, 1997; Revised Manuscript Received December 8, 1998
We analyzed the binding of heparinoid or heparin with fibrinogen by real-time measurement using surface plasmon resonance technology. Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate] and dextran sulfate were used as heparinoids. The binding ability of each sulfated polymer was estimated by having each polymer-containing buffer interact with the sensor chip surfaces that had immobilized fibrinogen. Dextran sulfate and poly(GEMA) sulfate showed high affinity to the fibrinogen in this experiment, while the heparin did not. All of the dextran sulfates were desorbed from its surface, while about 30% of the poly(GEMA) sulfate remained on the immobilized fibrinogen upon the addition of NaCl to the buffer which was done in order to analyze the desorption of poly(GEMA) sulfate or dextran sulfate from the surface of the fibrinogen. These data show that the type of binding between fibrinogen-poly(GEMA) sulfate was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was caused not only by an electrostatic but also by a hydrophobic force. These results suggest that the interaction mechanism of heparinoids with fibrinogen was different from that of heparin.
INTRODUCTION
Extensive research has been done about the interaction of blood plasma protein with polysaccharide sulfuric acid esters (1). Fibrinogen (which is blood clotting factor I), forms a soluble complex with polyanion [sulfate, polyvinyl sulfate, and polyisoprene sulfonate (2-6)], although fibrinogen contains an overall negative charge in a neutral buffer solution. The distribution of charges on a fibrinogen surface should be heterogeneous. This is a reasonable assumption based on the recently elucidated first-order structure and higher order structures of human fibrinogen (7, 8). In our previous study on glycoside polymer, we found that sulfated poly(glucosyloxyethyl methacrylate) [poly(GEMA) sulfate], which bears sulfated D-(+)-glucose, exhibited anticoagulant activity (9), and we studied their anticoagulant mechanism by evaluating various in vitro clotting tests (10, 11). Next, we found that poly(GEMA) sulfate and dextran sulfate formed insoluble complexes with bovine plasma fibrinogen by the turbidity measurement of each complex that contained PBS by using an UV-vis spectroscopy. In our experiment, the amount of poly(GEMA) sulfatefibrinogen insoluble complex increased with an increase in the amount of poly(GEMA) sulfate. On the other hand, the amount of dextran sulfate-fibrinogen insoluble complex showed a maximum (0.01 mg of dextran sulfate to 4.5 mg of fibrinogen in 1 mL of PBS) when dextran sulfate was added to fibrinogen PBS solution. However, * To whom correspondence should be addressed. Phone: +81992-85-8320. Fax: +81-992-55-1229. E-mail:
[email protected]. kagoshima-u.ac.jp.
these tendencies were not shown upon the addition of heparin, although heparin has a relatively high charge density (1.5-2.0/pyranose ring). The effects of the inhibition of fibrinogen polymerization by sulfated polymers act on both stages when A and B chains are split by thrombin and on the polymerization of fibrin monomers (3). Therefore, we concluded that poly(GEMA) sulfate or dextran sulfate formed insoluble complexes with fibrinogen and then these sulfated polymers appeared to have anticoagulant activity. Thus far, researchers have clarified the complex formation between fibrinogen and sulfated polymers by precipitation measurements, transmittance measurements using UV-vis spectroscopy, circular dichroism measurements, and electrophoretic mobility measurements with a Tiselius apparatus (2, 3, 5, 12). However, there have been few studies about kinetic measurements of these complex formations because it is very difficult to use the radio-labeling and fluorescence-labeling techniques (13) for either fibrinogen or sulfated polymers. Moreover, there has been no research about association and dissociation rate constant measurement of interaction between fibrinogen and sulfated polymer in situ. Therefore, a kinetic study on heparinoid-plasma protein interaction would provide significant data in regards to its anticoagulant activity. In this paper, we analyzed the interactions between plasma fibrinogen and each sulfated polymer at physiological pH values in detail using surface plasmon resonance (SPR) technology (14, 15). The BIAcore system (16-18) used in this study was produced by Pharmacia Biosensor AB (Uppsala, Sweden). BIAcore is a new type of technology that is designed to analyze label-free
10.1021/bc970218j CCC: $18.00 © 1999 American Chemical Society Published on Web 03/27/1999
Technical Notes
Bioconjugate Chem., Vol. 10, No. 3, 1999 539
Figure 1. Structure of poly(GEMA) sulfate (a), dextran sulfate (b), and heparin (c).
macromolecular interactions in real-time. The interactions between an immobilized molecule on a sensor chip and ligand is monitored by surface plasmon resonance, which detects changes in mass. We also studied the interactions of fibrinogen or albumin with some sulfated polymers for comparative process. The addition of salt to the buffer for desorption of each sulfated polymer from the immobilized fibrinogen gave insights into the mechanistic aspects of the interactions between each sulfated polymer and fibrinogen. MATERIALS AND METHODS
Materials. Poly(GEMA) sulfate was obtained by the sulfation of poly(GEMA) (9). We estimated the degree of sulfation of the poly(GEMA) sulfate by elemental analysis (which is the number of substituted hydroxyl groups in a glucose unit) to be 3.8. Heparin sodium salt (LotM2E) was purchased from Nacalai Tesque (Kyoto, Japan) and had an anticoagulant activity of 187.5 IU mg-1 solid. Dextran sulfate was also purchased from Nacalai Tesque and the degree of sulfation was 2.1. The structures of these sulfated polymers are shown in Figure 1. Bovine plasma fibrinogen and bovine serum albumin were purchased from Sigma (St. Louis, MO). A BIAcore, Sensor Chip CM5 (a carboxymethylated dextran that is coupled to a gold-coated glass surface according to procedures described elsewhere) and an Amine Coupling Kit containing EDC, NHS, and a solution of 1 M ethanol amine hydrochloride that was adjusted to a pH of 8.5 with sodium hydroxide were obtained from Pharmacia Diagnostics AB. All other reagents used were of analytical grade. In addition, MilliQ-grade water was used. SPR Apparatus. SPR phenomenon was first developed by Otto (14) and Kretschmann and Raether (15). A laser beam is directed onto the underside of a glass prism, which is an index that is matched to a metal-coated microscope slide and undergoes the total internal reflection at the glass/metal interface. A specific angle of incident beam and surface plasmon at the metal/air interface causes the surface plasmon to be excited and a leads to a subsequent reduction in the intensity of the internally the reflected light. The angle at which this excitation occurs (the SPR angle) is dependent upon the dielectric properties of the layers above the gold surface; therefore, SPR can be used to detect the adsorption of molecules at this interface. In an automatic instrument, BIAcore 2000, the sensor device is composed of three parts: a sensor chip that is a thin film of gold covered with carboxymethylated dextran, a prism which is placed on the opposite glass surface of the chip, and a microfluidices cartridge that can provide proper reaction fluid onto the surface of the sensor chip. As molecules adsorb
to the sensor surface, the change in the SPR angle is recorded as a function of time in order to observe the adsorption process onto the sensor surface in real time. Immobilization of Fibrinogen and Albumin. The immobilization of fibrinogen or albumin on Sensor Chip CM5 in BIAcore was done as follows (19, 20). Separate vials that contained 200 µL of 0.1 M NHS, 0.4 M EDC, and a 1 M ethanolamine hydrochloride solution and a vial for mixing EDC and NHS were placed in an autosampler together with a solution of fibrinogen or albumin that was to be immobilized. A continuous flow pump was filled with PBS buffer (pH 7.4). After Sensor chip CM5 had been conditioned with PBS for a few minutes, an automated immobilization cycle was performed at a flow rate of 5 µL min-1. Eighty microliters of EDC and NHS mix (1/1) was transferred to the mixing vial, and forty microliters of the mixed solution was injected on the surface. Fibrinogen and albumin were dissolved in a 10 mM acetate buffer (pH 5.0) at a concentration of 250 and 400 µg mL-1, respectively. Eighty microliters of each protein solution were then injected, followed by 35 µL of ethanolamine hydrochloride solution. Approximately 1.6 × 104 resonance units (RU) of fibrinogen and 1.2 × 104 RU of albumin were immobilized on the sensor chip. These corresponded to 4.7 × 10-5 nmol mm-2 and 17.8 × 10-5 nmol mm-2, respectively, based on the relationship in which 1000 RU equals 1 ng mm-2 bound protein (refractive index in protein ) 1.6). Interaction of Sulfated Polymers with Fibrinogen Surface. We estimated the binding of each sulfated polymer to the immobilized fibrinogen as follows: a sensor chip (CM5) with immobilized fibrinogen was brought into contact with the PBS buffer. The flow rate of the buffer solution was kept at a high level (10 µL min-1) in order to prevent mass transport limitation at the sensor surface/solution interface. After reaching a stable baseline (80 s), we injected 40 µL of PBS buffer which contained every sulfated polymer (0.3, 1.5, 3, and 30 µg mL-1), through the fibrinogen surfaces in order to observe the association rate constant (80-320 s). Next, we injected 40 µL of PBS buffer for the purpose of observing the dissociation rate constant (320-560 s). We monitored the changes in the resonance angle consecutively with time intervals of 1 s. This was done for 1520 min. Desorptions of Sulfated Polymers from Fibrinogen Surface. We estimated the degree of dissociation of poly(GEMA) sulfates or dextran sulfates from the fibrinogen surface by the addition of NaCl (0.03-3.0 M) in the PBS buffer. After performing the dissociation cycle (560 s, the resonance unit of this point is approximately
540 Bioconjugate Chem., Vol. 10, No. 3, 1999
Sakamoto et al.
Figure 2. The sensorgram showing the procedure and response of the immobilization of fibrinogen to the sensorchip surface. The letters A, B, C, and D indicate time intervals during which the sensor was in contact with different solutions (A, PBS buffer; B, EDC/NHS; C, acetate buffer with fibrinogen 250 µg mL-1; D, ethanolamine hydrochloride).
constant), we injected NaCl-containing buffer (10 µL) into each of the heparinoid-fibrinogen complex surfaces and the remaining heparinoid on the fibrinogen surface (which was represented by a resonance unit) was estimated as a ∆RU560s/∆RU930s ratio (Figure 6a). RESULTS AND DISCUSSION
Interactions of Sulfated Polymers with Protein Surface. Figure 2 shows a sensorgram that was recorded during the immobilization of fibrinogen onto the carboxymethylated dextran that covered the sensorchip of a plasmon resonance sensor. The SPR angle is represented as a resonance unit (RU) in BIAcore (0.1° ) 1000 RU). During the time interval when the sensor was in contact with the fibrinogen solution, we observed an increase in the resonance unit (RU), which indicates the adsorption of fibrinogen onto the surface. The shape of the response curve (C) indicates that the saturation of the surface with fibrinogen is slow. The RU (after the immobilization of the fibrinogen) was normalized to D as the baseline. If sulfated polymers are injected, we can monitor the time course of the binding as a change in RU. The sensorgram gives data about the association of the complex that can be followed; the equilibrium or the steady state is readily displaced by the analyte sulfated polymer flow. When the analyte is replaced by a buffer flow, the dissociation of the complex is also monitored. In this study, bovine plasma fibrinogen was covalently coupled to the carboxymethylated dextran surface of the sensorchip as a ligand and sulfated polymer (analyte) buffer solution was flowed. This is because we detect nonspecific adsorbed albumin and fibrinogen on the carboxymethylated dextran surface and we did not detect any adsorbed all sulfated polymers on its surface in the control experiments. The overlay sensorgrams for the interaction of each sulfated polymer with fibrinogen are shown in Figure 3. We collected binding curves from different batches of proteins because fibrinogen was an unstable protein and tended to denature in the regeneration process. We observed an increase in RU during its contact with a continuous flow (10 µL min-1) of the PBS buffer that contained poly(GEMA) sulfate or dextran sulfate at a concentration of 0.3-30 µg mL-1, indicating a net ad-
Figure 3. Adsorption of poly(GEMA) sulfate (a), dextran sulfate (b), and heparin (c) to immobilized fibrinogen. Each sulfated polymer concentration is 0.3, 1.5, 3, and 30 µg mL-1. PBS buffer, pH 7.4.
sorption of each heparinoid to the immobilized fibrinogen. During subsequent contact with the PBS buffer, the RU decreased slightly, indicating a net desorption of each heparinoid from the fibrinogen (Figure 3, panels a and b). On the other hand, its adsorption of heparin was slightly observed as a refractive index shift (Figure 3c), which indicates its lower affinity for heparin. These results clarified that poly(GEMA) sulfate and dextran sulfate directly formed complexes with fibrinogen; heparin, however, did not. These results are also consistent with our previous research (10). Using the sensorgrams for the binding of poly(GEMA) sulfates and dextran sulfates with fibrinogen (Figure 3, panels a and b) we were able to obtain the maximum binding RU (∆RUmax) of each heparinoid on the fibrinogen surface at a suitable concentration as a function of each
Technical Notes
Bioconjugate Chem., Vol. 10, No. 3, 1999 541
Figure 4. Adsorption isotherms for poly(GEMA) sulfatebinding (O) and dextran sulfate-binding (0) to fibrinogen surfaces. PBS buffer, pH 7.4.
heparinoid concentration (Figure 4). The ∆RUmax curves for poly(GEMA) sulfate and dextran sulfate on fibrinogen reached plateaus at a high concentration of heparinoid (30 µg mL-1), indicating that the adsorption behavior of each heparinoid on the fibrinogen was Langmuirian. This suggests that there was neither multilayer aggregation nor heterogeneous adsorption on the fibrinogen surface. The SPR method can directly evaluate the timedependent interaction between heparinoid and fibrinogen; the turbidity measurement of the heparinoidfibrinogen complex, however, cannot. Therefore, association and dissociation processes can be monitored by the SPR method, providing a base for kinetic studies. The time needed to reach an equilibrium state for the binding of each heparinoid (30 µg mL-1) to the fibrinogen surface was about 5 min (Figure 3, panels a and b). In our previous study (10), we evaluated the turbidity of the heparinoid-fibrinogen mixture solution in a PBS buffer by allowing a heparinoid-fibrinogen mixture solution to stand for 10 min. The SPR results described above show that the turbidity measurements were done in an equilibrium state. However, another advantage of SPR measurements in regard to analyzing the direct affinity between heparinoid and fibrinogen is because turbidity measurements can only detect large aggregated structures, although the latter is much easier to measure than the former. We calculated the dissociation constant (KD) from sensorgrams for the binding of each heparinoid to fibrinogen (Figure 3, panels a and b) by following pseudofirst-order kinetics (BIAcore system manual). We obtained very high correlation coefficients in the determination of KD for the binding of both heparinoids (∼0.99), and a KD (6.2 × 10-10 M) for the binding of dextran sulfate to fibrinogen was lower than that (3.1 × 10-8 M) for poly(GEMA) sulfate. This might be due to differences in electrostatic force between dextran sulfate and poly(GEMA) sulfate. In this study, we did not confirm the presence of a stoichiometrical complex because the refractive index of poly(GEMA) sulfate and dextran sulfate have not been obtained by anyone yet. If refractive indices of heparinoids are obtained, the interaction mechanism between each heparinoid and fibrinogen can be analyzed in more detail. The overlay sensorgrams for the interaction of each sulfated polymer with albumin are also shown in Figure 5. We observed lower nonspecific adsorption of all sulfated polymers to the immobilized albumin as a slight refractive index shift. These results indicate the lower
Figure 5. Adsorption of poly(GEMA) sulfate (a), dextran sulfate (b), and heparin (c) to immobilized albumin. PBS buffer, pH 7.4.
affinity of all sulfated polymers for the albumin. However, it seems that the BIAcore system is not able to detect the binding profiles of a lower association constant (KD < 104 M). In addition, each sulfated polymer may be prevented from penetrating the albumin-immobilized matrix by steric factors. Poly(GEMA) sulfate and dextran sulfate showed a high affinity with fibrinogen in this study, while heparin had little or no affinity. The binding amount was dependent on the heparinoid. These results suggest that the interaction mechanism of heparinoid with fibrinogen was different from that of heparin. It is well-known that the aggregation of plasma fibrinogen at physiological pH values is caused by the addition of dextran sulfate, although no aggregations have been observed upon addition of heparin, because of the limited number of
542 Bioconjugate Chem., Vol. 10, No. 3, 1999
Sakamoto et al.
Figure 7. Schematic representation of interaction between each heparinoid and fibrinogen.
Figure 6. (a) The sensorgram showing the effect of the addition of the 1.0 M NaCl on desorption of poly(GEMA) sulfate from immobilized fibrinogen. (b) Decrease of ∆RU560s/∆RU930s by the desorption of poly(GEMA) sulfate (O) and dextran sulfate (0) from the fibrinogen surface with the addition of NaCl containing PBS buffer.
association constants between heparin and fibrinogen. Therefore, these results are consistent with the above. Desorption of Sulfated Polymer from Fibrinogen Surface. To obtain data on the binding mechanism of poly(GEMA) sulfate or dextran sulfate to fibrinogen, we examined the degree of dissociation of a sulfated polymer from immobilized fibrinogen in the presence of a NaClcontaining PBS buffer. In general, the interaction of protein with other molecules is believed to be due to (i) ion-ion interaction, (ii) ion-dipole and dipole-dipole interaction, (iii) solvophobic interaction, and (iv) dispersion or van der Waals forces. Our experiment will show if an insoluble complex formation will only lead to an electrostatic interaction between heparinoid and fibrinogen. A sensorgram of the poly(GEMA) sulfate desorption from immobilized fibrinogen with a PBS buffer that contains 1.0 M NaCl is shown in Figure 6a. ∆RU560s indicates the amount of binded heparinoid to the fibrinogen surface after 560 s from the starting point of the experiment. ∆RU930s (RU930s can be seen as a stable baseline because dissociation curve in Figure 6a represents very slow kinetics) indicates the amount of remaining heparinoid on the sensor chips after the injection of NaCl containing buffer (0.03-3.0 M) for 330s to the heparinoid-fibrinogen complexes. We compared the ∆RU560s/∆RU930s values for poly(GEMA) sulfate against NaCl concentration with those for dextran sulfate (Figure 6b). The amount of adsorbed heparinoid to fibrinogen surface decreased with an increase in the ionic strength of the NaCl-containing buffer. Upon the addition of a 1.0 M NaCl containing buffer, all of the dextran sulfates were
desorbed from it, while the poly(GEMA) sulfates were incompletely desorbed, about 30% of poly(GEMA) sulfate remained on the fibrinogen surface despite the presence of a 2.0 M NaCl containing buffer injection. Above concentrations of 2.0 M, we also observed the higher adsorption of both heparinoids to fibrinogen. A consideration of above result can be explained by the denaturing of immobilized fibrinogen. These data show that the binding behavior of poly(GEMA) sulfate with fibrinogen was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was not only caused by electrostatic but also by hydrophobic force (Figure 7). This is because the poly(methacrylate) backbone of poly(GEMA) sulfate is the most hydrophobic part in the polymer. In our previous study, we noted that the interaction mechanism of poly(GEMA) sulfate to fibrinogen was differed from that for dextran sulfate (10). This is consistent with our previous research. In further research, we will clarify the formation mechanism of heparinoid-fibrinogen complexes in detail by using different kinds of salt, i.e., by using a lyotropic series that is a defined as the salting-out effects on ions on protein as well as the effects of ions on the surface tension of water (21). The interactions between fibrinogen and sulfated polymers could be observed using the SPR technique in real-time. This is consistent with our turbidity measurement study (10). The kinetic constants between heparinoid-fibrinogen can also be obtained. We clarified the differences in the interaction mechanism of poly(GEMA) sulfate to fibrinogen and that of dextran sulfate by using a NaCl-containing buffer. In general, SPR methodology is used for the kinetic determination of the interaction of macromolecules. In this study, we showed that these SPR methods and experimental procedures can be used to obtain data on mechanistic interactions between various sulfated polymers and various plasma proteins by using a salt-containing buffer. This methodology cannot only obtain the kinetic constants between antigenantibody but also gives us new and valuable insights into physiological mechanistic aspects such as the interactions between body components (proteins, phospholipids, DNAs, and cells) and biomaterials (natural occurring polymers, synthetic polymers, and inorganic materials). ACKNOWLEDGMENT
This study was financially supported in part by the project entitled “High and Ecological Utilization of Regional Carbohydrates”, through Special Coordination Funds for Promoting Science and Technology (Leading
Technical Notes
Research Utilizing Potential of Regional Science and Technology) of the Science and Technology Agency of the Japanese Government, 1997, and this study also was financially supported in part by a Grant-in-Aid for Scientific Research in Priority Areas of “Super-Biosystem Constructed by Cognitive Multidimensional Glyco-molecules” (no. 285/09240105) from the Ministry of Education, Science, Sports and Culture, Japan. The authors thank Dr. Akio Kishida of Kagoshima University for their experimental supports and helpful discussions. LITERATURE CITED (1) Gorter, E., and Nanninga, L. (1953) Complexes of heparin and protein. Discuss. Faraday Soc. 13, 205-217. (2) Astrup, T., and Piper, J. (1946) Interaction between fibrinogen and polysaccharide polysulfuric acids. Acta Physiol. Scand. 11, 211-220. (3) Sato, H., Nakanishi, E., and Nakajima, A. (1981) Electrochemical and conformational study of the interactions of fibrinogen with acidic polysaccharides. Int. J. Biol. Macromol. 3, 66-70. (4) Walton, K. M. (1952) The biological behavior of a new synthetic anticoagulant (dextran sulphate) possessing heparin-like properties. Brit. J. Pharmacol. 7, 370-391. (5) Sasaki, S., and Noguchi, H. (1959) Interaction of fibrinogen with dextran sulfate. J. Gen. Physiol. 43, 1-12. (6) Tamada, Y., Makino, K., Yoshida, T., and Taniguchi, Y. (1996) Blood chemical analysis. G. Development of a new blood anticoagulant. Rinsho-Byori (Japanese) 103, 192-199. (7) Doolittle, R. F., Watt, K. W. K., Cottrell, B. A., Strong, D. D., and Riley, M. (1979) The amino acid sequence of the a-chain of human fibringen. Nature 280, 464-468. (8) Doolittle, R. F., Everse, S. J., and Spraggon, G. (1996) Human fibrinogen: anticipationg a 3-dimensional structure. FASEB J. 10, 1464-1470. (9) Akashi, M., Sakamoto, N., Suzuki, K., and Kishida, A. (1996) Synthesis and anticoagulant activity of sulfated glucosidebearing polymer. Bioconjugate Chem. 7, 393-395. (10) Sakamoto, N., Kishida, A., Maruyama, I., and Akashi, M. (1997) The mechanism of anticoagulant activity of a novel heparinoid sulfated glucoside-bearing polymer. J. Biomater. Sci. Polym. Ed. 8, 545-553. (11) Ohnishi, M., Miyashita, Y., Motomura, T., Sakamoto, N., and Akashi, M. Anticoagulant and antiprotease activities of
Bioconjugate Chem., Vol. 10, No. 3, 1999 543 a heparinoid sulfated glucoside-bearing polymer. J. Biomater. Sci. Polym. Ed. 9, 973-984. (12) Nakajima, E., Sato, H., and Nakajima, A. (1990) Electrophoretic study on a soluble complex of heparin and fibrinogen. Polym. J. 22, 510-517. (13) Hlady, V., Van-Wagenen, R. A., and Andrade, J. D. (1985) Total internal reflection intrinsic fluorescence (TIRIF) spectroscopy applied to protein adsorption. in Surface and Interfacial Aspects of Biomedical Polymers (J. D. Andrade, Ed.) Vol. 2, pp 81-119 Plenum Press, New York. (14) Otto, A. (1968) Excitation of nonradiative surface plasmon waves in silver by the method of frustrated total reflection. Z. Phys. 216, 398-410. (15) Kretschmann, E., and Raether, H. (1968) Radiative decay of nonradiative surface plasmons excited by light. Z. Naturforsch. 23, 2135. (16) Karlsson, R., Michaelsson, A., and Mattsson, L. (1991) Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J. Immunol. Methods 145, 229-240. (17) Chaiken, I., Rose´, S., and Karlsson, R. (1992) Analysis of macromolecular interactions using immobilized ligands. Anal. Biochem. 201, 197-210. (18) Altschuh, D., Dubs, M. C., Weiss, E., Zeder-Lutz, G., and Van-Regenmortal, M. H. V. (1992) Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance. Biochemistry, 31 6298-6304. (19) Johnsson, B, Lo¨fås, S., and Lindqquist, G. (1991) Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal. Biochem. 198, 268-277. (20) Huber, W., Hurst, J., Schlatter, D., Barner, R., Hu¨bscher, J., Kouns, W. C., and Steiner, B. (1995) Determination of kinetic constants for the interaction between the platelet glycoprotein Iib-IIIa and fibrinogen by means of surface plasmon resonance. Eur. J. Biochem. 227, 647-656. (21) Melander, W., and Horva´th, C. (1977) Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch. Biochem. Biophys. 183, 200-215.
BC970218J
