BS133_SPR_4A

4. Equilibrium constants : 

4. Equilibrium constants 4.1 Surface plasmon resonance (SPR): An instrument to measure binding   The SPR (biacore) instrument is used to measure binding constants, K, between 2 proteins or between a protein and a ligand. Disposable sensor chip Ligand, , is covalently coupled to a hydrophobic/ hydophilic matrix, e.g. carboxylated dextran surface coupled to thin gold film.   Macromolecule, , injected through flow cell - repeat at a series of concentrations. Why? (Hint - this is like a kinetics experiment) About 10 readings can be taken per second so data is recorded in real time.

4.1 SPR: An instrument to measure binding : 

4.1 SPR: An instrument to measure binding If macromolecule binds to ligand, increase in mass in vicinity of the sensor is detected as a change in refractive index. (You will measure refractive index in the lab.) Change in refractive index is detected using optical technique based on surface plasmon resonance. (Surface plasmon resonance is a quantum mechanical phenomena.) It is detected as light extinction at a special angle - this angle depends on the refractive index near the surface. The surface plasmon resonance occurs in the gold film. Many biochemical techniques for studying interactions between proteins or proteins and ligands rely on coupling another molecule, e.g. a probe fluorescent molecule, and looking at changes in fluorescence. This is a label free technique for monitoring interactions. The advantage of a label free technique is… Volume used is ~60 nL. The small volume and high sensitivity makes it suitable for fragment-based drug design (see BS317).

4.1 SPR: An instrument to measure binding : 

4.1 SPR: An instrument to measure binding The ligand binding response is measured in response units, RU. 100 RU corresponds to a particular protein load measured in g mm-2. Typical results are shown below Response /RU Time/s Binding constant, K is given by K = dissociation association konis rate const for binding; koff for dissociation

4.1 SPR: An instrument to measure binding : 

4.1 SPR: An instrument to measure binding If you can measure K, how would you obtain:   ·       G ·       H ·       S   In reality, complex mathematical analysis using sophisticated computer programs is required to determine binding data using SPR. That will not stop you using the technique professionally.   The BIACORE SPR instrument is available from Pharmacia Biosensors, Uppsala, Sweden. Other manufacturers sell similar instruments but this one is probably the best in 1999. Reference, Current opinion in Biotechnology 9, 1998, 97-101; this is not is the Essex University Library - but you will not be examined on BIACORE/SPR in BS133. G° = -RTlnK Plot lnK against 1/T, gradient gives H° G = H - TS 10