2. CHAPTER 2 – Materials and Methods
2.20. Surface Plasmon Resonance
2.20.2. Sensor chips
The sensor chips purchased from BIAcore AB, consist of a glass surface, coated with a thin layer (~50 nm) of gold. A range of specialised surfaces are available, allowing the immobilisation of a variety of molecules.
The most widely used sensor surface, consists of the gold surface modified with a carboxymethylated dextran matrix, 100-200 nm thick (Stenberg et al 1991, http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf, accessed December 2002). This dextran matrix can be derivatised to allow a variety of immobilisation chemistries. The dextran layer provides a hydrophilic environment to preserve biomolecules in a native state. It also increases the volume of the surface increasing the potential level of ligand immobilisation and hence the sensitivity of the
O O O O O O
Gold surface
O O N H O C H2 C H2 OH NH O TolBGold surface
Activation with EDC/NHSActive esters react with NH2groups on TolB Deactivation of
remaining esters with ethanolamine
Gold surface
O O N O O O O N O O O OGold surface
O O NH O N O O O O TolB O O O O O OGold surface
O O O O O OGold surface
O O N H O C H2 C H2 OH NH O TolBGold surface
O O N H O C H2 C H2 OH NH O TolBGold surface
Activation with EDC/NHSActive esters react with NH2groups on TolB Deactivation of
remaining esters with ethanolamine
Gold surface
O O N O O O O N O O O OGold surface
O O N O O O O N O O O OGold surface
O O NH O N O O O O TolBGold surface
O O NH O N O O O O TolBFigure 2.14 Amine coupling TolB protein to the surface of a CM5 chip (Biacore AB).
2.20.3.
Amine coupling
A widely applied immobilisation procedure of the carboxymethylated dextran matrix activates the carboxyl groups of the matrix using a mixture of EDC and NHS, via the same chemistry shown in Figure 2.10, to form esters that can react with amine groups on proteins, resulting in covalent immobilisation of the protein to the surface (see Figure 2.14). The protein must be diluted in buffer at a pH lower than the pI value of the protein to allow electrostatic attraction of the protein to the negatively charged matrix. Caution must be taken when using this method because the low pH used has the potential to denature the protein and therefore the activity of the protein should be measured after coupling. Another disadvantage of this method is that the protein is randomly orientated on the sensor chip surface due to coupling of different lysine residues to the matrix, which may introduce sub-populations of accessibilities and reactivities (O’Shannessy et al 1992)
A variety of other immobilisation methods have been described (O’Shannessy et al 1992), for example, coupling by thiol/disulfide exchange [Cunningham and Wells 1993, O’Shannessy et al 1992). Other surfaces have also become available from BIAcore AB including surfaces functionalised with NTA, for capture of ligands via metal chelation; streptavidin, for capture of biotinylated ligands; and flat, hydrophobic surfaces to allow deposition of liposomes on the surface.
2.20.4.
Microfluidics
Biacore systems are equipped with a microfluidics system that allows analyte to pass over the sensor surface in a continuous, pulse-free and controlled flow
(www.biacore.com technology brochure). This allows the concentration at the sensor surface to remain constant.
2.20.5.
Reference cell
The microfludics system allows at least two channels to be measured simultaneously. This allows comparison of binding between a derivatised surface and a blank
reference surface. Binding to the reference surface can be automatically subtracted from the binding measured on the derivatised surface, so that the response recorded is due to specific binding of the analyte to the sample surface.
2.20.6.
Regeneration
Following an analyte injection, remaining analyte bound to the ligand must be removed from the sensor surface, with the ligand remaining intact, before another analyte injection is performed ie the surface must be regenerated. The choice of regeneration conditions is dictated by the nature of the ligand-analyte interaction, the stability of the ligand and the type of ligand immobilisation employed. For example for weak interactions, an increased speed of buffer flow may remove remaining analyte. Other conditions that can be used are high or low pH (eg 10 mM glycine pH 2, 50 mM NaOH), high salt (eg 1-2 M NaCl) or detergent (eg SDS). The most frequent method used is injection of 10 mM glycine pH 1.5-2.5. This pH change causes most proteins to partially unfold and become positively charged. The protein binding sites will therefore repel each other, moving the ligand and analyte further apart. Regeneration conditions must be optimised to ensure that the ligand remains intact and fully functional after the regeneration step.
2.20.7.
Applications
SPR can be used in a number of ways to characterise biomolecular interactions. SPR can provide quantitative information on affinity, kinetics, specificity and the effect of different conditions eg buffers, co-factors, temperature etc on complex formation.
2.20.7.1. Affinity
Affinity is generally expressed as either the association constant (Ka), expressed in units of M-1 or the dissociation constant (Kd), which is the inverse of Ka and hence has units of M.
For the interaction A + B ↔ AB,
Ka and Kd can be measured directly by equilibrium binding analysis or calculated from the rate of association (kass) and rate of dissociation (kdiss).
Affinity constants can be measured by using equilibrium binding analysis on the BIAcore instrument. This involves injection of a series of analyte concentrations and measurement of the response at equilibrium. In most cases, the data produced can then be fitted to the Langmuir binding model to derive affinity constants (see Equation 2-10). The model assumes that the analyte is monovalent and
homogeneous, that the ligand is homogeneous and that all events are independent.
Equation 2-10 The Langmuir binding isotherm, where Req is the response measured at equilibrium and Rmax is the maximum response at complete saturation of immobilised binding sites
A Scatchard plot of the same data, obtained by plotting Req/[A] against Req, can be used to visualise the extent to which the data fit the model (BIAevaluation Software Handbook). A linear Scatchard plot is consistent with the data fitting the model. However, Scatchard plots alone should not be used to estimate affinity constants because they place inappropriate weighting on the data obtained with the lower concentrations of analyte, which are generally the least reliable
(http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf, accessed December 2002).
2.20.7.2. Kinetic analysis
The real-time binding data collected using SPR allows the rate constants for an [A] * Rmax [A] + Kd Req = [AB] [A] [B] Ka =
SPR measures the kinetics of binding to surface-immobilised ligand under conditions of continuous flow with the concentration of free analyte, [A], kept constant by continuous supply of new sample. The concentration of complex on the surface, [AB], is given by the response above baseline.
Although the concentration of free ligand on the surface [B] is not measured directly, the total ligand concentration can be expressed in resonance units as the maximum analyte binding capacity ie Rmax and the concentration of free ligand is then Rmax – [AB].
d[AB]
dt = kass[A][B] – kdiss[AB] d[AB]
dt = kass[A][B] – kdiss[AB]
Equation 2-11 General equation for binding kinetics
After the sample has passed over the surface, analyte that dissociates from the complex is carried away by the buffer flow, so that the free analyte concentration is zero. The rate of dissociation is then described by Equation 2-12.
[AB] = dt - kdiss[AB] [AB] = dt - kdiss[AB] Equation 2-12
Therefore, the rates of association and dissociation can be determined from Equation 2-11 and Equation 2-12.
There are important limitations to kinetic analysis using SPR: 1. Mass transport
This effect can occur with high ligand densities where the rate at which the surface binds the analyte exceeds the rate at which the analyte can be delivered to the surface. Therefore the measured association rate (kass) is slower than the true kass. Analyte is transported to the surface by diffusion and convection. Increasing the flow rate can increase convection transport and diffusion limitations can be reduced by decreasing
2. Rebinding of analyte
If, following dissociation of analyte, the analyte rebinds to unoccupied ligand before diffusing away from the surface, the measured dissociation rate (kdiss) will be slower than the true kdiss. Rebinding can be reduced by increasing the flow rate, thus increasing convection transport to remove the analyte at a faster rate and by decreasing the level of ligand immobilisation.
These limitations mean that it is difficult to measure kass values faster than approximately 106 M-1 s -1 and kdiss values slower than 10-5 s-1 or faster than 1 s-1 (http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf, accessed December 2002).
2.20.8.
SPR Methodology
SPR was performed using BIAcore J and BIAcore X instruments from BIAcore AB (Uppsala, Sweden), operating BIAcore J and BIAcore X control software
respectively. Operation and maintenance procedures were carried out as detailed in the BIAcore J and BIAcore X instrument handbooks, using BIAcore AB certified products. HBS-EP running buffer (10mM Hepes [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% [v/v] surfactant P20), purchased from BIAcore AB, was used throughout. All buffers and solutions used were filtered and degassed using 0.2 µm sterile syringe filters (Millipore) or Steritop sterile bottle-top filters (Millipore). Solutions filtered by syringe filter were degassed by centrifugation.
2.20.8.1. Immobilisation of proteins on CM5 sensor chips
The ligand was immobilised to the matrix of a newly docked CM5 sensor chip, pre- equilibrated in HBS-EP running buffer (BIAcore AB) via amine coupling. Medium flow rate (~ 35 l min-1) was used in the BIAcore J instrument and a 10 l min-1 flow rate was used in the BIAcore X instrument throughout ligand immobilisation. The carboxymethylated matrix in both flow cells was activated using a mixture of 0.1 M ethyl-N-(3-diethylaminopropyl) carbodiimide (EDS, BIAcore AB) and 0.4 M N- hydroxysuccinimide (NHS, BIAcore AB). The protein sample was diluted in coupling buffer (BIAcore AB, 10 mM sodium acetate, pH 4.5)and injected in single channel mode across flow cell 2 until the required amount of protein was immobilised on the surface. Remaining active esters in both flow cells were then deactivated using
The final level of immobilisation was determined after two washes with HBS-EP buffer. A response of 1000 RU equates to 1 ng of protein attached to the chip (www.biacore.com/technology/spr_technology.lasso).
2.20.8.2. SPR binding analysis
Measurement of binding of proteins to the immobilised protein on the CM5 sensor chip was completed at 25 ºC with HBS-EP running buffer using medium flow rate (BIAcore J) or 30 l min -1 (BIAcore X). Proteins to be used as analytes were diluted in HBS-EP buffer to the required concentration and a two minute injection of the diluted protein was performed. The surface was then regenerated using a two minute injection of 10 mM glycine (pH 1.8). The experiment was repeated twice for each concentration of protein.
2.20.8.3. SPR data analysis
Data from the BIAcore J instrument were analysed using BIAviewer Software (BIAcore AB).
All data used for kinetic analysis were obtained using the BIAcore X instrument. Kinetic analysis for binding of analyte to the immobilised ligand was achieved using BIAevaluation software version 3.1. Global analysis was performed employing the Langmuir 1:1 binding isotherm where very rapid changes in response are accounted for as refractive index changes.