Transient kinetic methods. Biophysics seminars Kinga Futó

Transcription

1 Transient kinetic methods Biophysics seminars Kinga Futó

2 Fast kinetics Reaction kinetics: temporal description of reactions reactionmechanisms reaction dynamics (processes on molecular level) rate of reaction = velocity of a reaction: stoichiometric coefficient molar number

3 Role of rate of the reaction Rate depends on: quality of materials and molecules concentrations temperature catalisators / inhibitors reaction orders (Not stoichiometric coeff.!) a+b: gross reaction order

4 Concentration-dependence of the rate of the reaction

5 Molecularity Unimolecular reaction: alteration of a single molecule. Bimolecular reaction: collision of two molecules. Trimolecular reaction: rare, because more then 2 molecules have to collide. PRIMARY REACTIONS can not be separated into simple steps. The order of primary reactions is determined by their molecularity.

6 Relation of order of process and molecularity Rate equation rate constant conc. of initial substrate conc. of product(s)

7 3%B3sebess%C3%A9g Reaction of 0 th order e.g. enzyme catalysis (if teh conc. of enzyme is low) NOT primary reaction!!! Initial substrate 1. t 1/2 Products 2. t 1/2 3. t 1/2 The concentration of components changes linearly in terms of time.

8 3%B3sebess%C3%A9g Reaction of 1 st order A B or A B + C Initial molecule 1. t 1/2 Products 2. t 1/2 3. t 1/2 The concentration of components changes exponentially in terms of time.

9 3%B3sebess%C3%A9g Reaction of 2 nd order 2A B (or A + B C) Initial molecule 1. t 1/2 Products 2. t 1/2 3. t 1/2 The concentration of components changes according to a hyperbolic equation in terms of time.

10 Complex reactions = reaction mechanisms Serial chemical reactions: rate limiting step: B C rate limiting step : A B Parallel chemical reactions: Equilibrium constant: Equilibrium chemical reactions:

11 Energetics For effective collision, molecules have to get in an active state (to have excess energy). The difference of the threshold energy and the average energy is the activation energy. initial state active state end state extent of reaction

12 Transient kinetic experiments understanding biological mechanisms time scale of biological processes measurements e.g. in ms range time-scale in reaction kinetics s ms µs ns ps mixing by hands stopped-flow flash photolysis photon counting Source: precentation of Ernő Keszei

13 Fast kinetic methods Stopped-flow Surface Plasmon Resonance Flash Photolysis Photon Counting

14 Stopped-flow

15 0. Filling cell mixer stop A B drive STOP sensor

16 1. Mixing drive STOP sensor

17 1. Mixing drive STOP sensor

18 1. Mixing drive STOP sensor

19 1. Mixing drive STOP sensor

20 2. Stopping the flow t = o drive STOP!

21 3. Kinetic measurement fluorescence or scattering absorption drive STOP sensor

22 Dead time The time elapsed between mixing and the start of data acquisition 0,5 1 ms Absorbance time (s)

23 Dead time time (s)

24 Forrás: Applied Photophysics Dead time

25 Applications of Stopped-flow Study of association- and dissociation kinetics in case of protein-protein, and protein-ligand interactions K D : dissociation constant K D = [ A][ B] [ AB]

26 The signal Spectroscopic signal has to change during reaction! fluorescence intensity (or anisotropy) light scattering absorption parallel detection! perpendicular detection! detector: PMT

27 Flash photolysis (Lysis of molecules due to the effect of light)

28 Flash photolysis Monitoring reactions of short life particles. Caged molecule: inactivated by a chemical bond. Freed by a high intensity light flash (inactivating bond is decomposed) process starts. MOlecules A and B are togedther (mixing does not take rime). Process starts when the light is applied. Detecting the light, the zero time of the spectroscopic measurement can be found.

29 Surface Plasmon Resonance (SPR)

30 metal plasmon Surface Plasmons prism Electrons moving in a wave-like manner on the surface of metals. Wavelength is shorter than that of exciting light. Far field: we can observe an illuminated object by watching the reflected light through a microscope or telescope. The electric and magnetic field of the light waves is equal in magnitude and interdependent. Near field: The component of the light that sticks to the surface The magnetic component is weak compared to the electric (no diffraction and interference occurs).

31 commons/7/78/spr-schema.png Plasmon generation Kretchmann configuration Dispersion relation: describes the relation between the energy and wavelength (impuse) of plasmons. Effective excitation: if the wavelengths or the impulses are equal (conservation of impulse). Wavelength of light in glass is shorter than in air. Impulse is greater. Total reflexion occurs on the prism-air boundary, but sticking to the the surface, the near field appears (with exponentially decreasing gradient with diverging distance).

32 The condition of the resonance The component of the exciting light impulse must be equal to the impulse of a surface plasmon of equal energy. Excitation occurs at an appropriate incident angle of light a minimum is observed in the intensity of the light refracted from the metal surface (absorption!).

33 The principle Flow chamber Partner B Metal (e.g. gold or silver) plate Glass Partner A Light Detection

34 The principle E Metal (e.g. gold or silver) plate Glass Dip (decrease) in intensity!

35 The sensogram the origin of a signal Metal (e.g. gold or silver) plate Glass I. II. Binding changes the reflected angle.

36 Detecting SPR The incident angle matching the resonance conditions is sensitively dependent on the refractive index of the prism. Any binding process on the surface changes the refr. index of the layer shift in resonanace angle sensitive detection. Analitic and kinetic information: temporal changes in resonance angle.

37 edia/commons/3/3d/sprcurve.png Affinity of two ligands Binding constant determination Equilibrium value of the product quotient: association rate divided by dissociation rate. Bait ligand is immobilized prey analyte is injected prey analyte binds bait ligand increase in SPR signal. After association: solution without the prey analyte injected dissociation decrease in SPR signal. From association and dissociation rates binding constant

38 A. Campion, P. Kambhampati, Chemical Society Reviews, 27, 241, (1998) Applications of SPR Antibody antigen DNA hybridisation Protein protein Receptor biomolecule interactions Determination of Kinetic constants and specificity of molecular interactions High sensitivity analytics