Kinetic & Affinity Analysis - IQM CSIC · 1/ GE / Kinetic & Affinity Analysis An introduction 2/ GE Biacore T100 training_kinetics/ What are kinetics and affinity? Kinetics • How

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Kinetic & Affinity Analysis

An introduction

2 / GE Biacore T100 training_kinetics /

What are kinetics and affinity?

Kinetics

• How fast do things happen? – Time-dependent

• Association – how fast molecules bind

• Dissociation – how fast complexes fall apart

• Kinetics determine whether a complex forms or dissociates within a

given time span

Affinity

• How strong is a complex? – Time-independent

• Affinity determines how much complex is formed at equilibrium (steady state where association balances dissociation)


What is the relevance of binding kinetics?

The cell is a dynamic system – rarely in equilibrium

The same affinity can be resolved into different on and off rates for

different interactions

• Kinetic data reveal more information

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Same affinity but different kinetics

All 4 compounds have the same affinity KD = 10 nM = 10-8 M

The binding kinetic constants vary by 4 orders of magnitude

kon koff (M-1s-1) (s-1)

106 10-2

105 10-3

104 10-4

103 10-5

Time

Concentration = 1000 nM

30 min

60 min

All target sites

occupied

Re

spo

nse

Time

30 min

60

min

Concentration = 100 nM


Three ways to obtain kinetic and affinity data in Biacore

Monitor association and dissociation rates

• Affinity Kinetics

Monitor steady state levels


Measure free analyte in solution



Rate equations for 1:1 kinetics

A

B

A

BA B

ka

kdAB

Association:

Dissociation:

Net rate equation:

where

ka-1

[s ]

-1 -1[M s ]

kd dissociation rate constant

association rate constant

kd [AB][AB]-ddt

ka[AB]ddt

[A] [B]

ka kd [AB][AB]d

dt[A] [B]

M/s M-1s-1s-1M M M

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Equilibrium constants

The equilibrium constants:

-1[M ]the constantequilibrium association

the dissociation constantequilibrium

ka

kd

kd

ka

[AB][A] [B]

[M][AB]

[A] [B]

At equilibrium:

Association Dissociation

kd [AB][A] [B]ka

s-1M s-1-1 M M M

KA

KD


Equilibrium and kinetics in Biacore

A is the analyte in solution

• Free concentration maintained constant by flow system

AB is the complex

• Concentration of complex measured directly as R in RU

B is the ligand on the surface

• Total concentration can be expressed in RU, as maximum binding capacity Rmax

• Free concentration is Rmax-R

A + B ⇔ AB

ka

kd

We do not need to know the “real” concentration

of ligand or complex


Rate and affinity in Biacore terms

A Bka

kdAB

ka kd [AB][AB]ddt

[A] [B]

RU/s M RU RU

dR

dtka kdmax R][R RC

AB

has one binding site and reacts with immobilized ligandhas n identical and independent binding sites

s-1M s-1-1

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The net rate equation terms in a sensorgram


Information in a Sensorgram

The relationship between Rmax, Req and KD


Mass transport

A phenomenon with relevance to kinetics measurements in

Biacore

• Describes the movement of molecules from solution to a surface

• Is independent of biomolecular interaction processes

Rates measured in Biacore depend on both mass transport and

biomolecular binding

• The relative importance of mass transport effects can be largely controlled by the assay conditions used

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What is mass transport?

Diffusive mass transport

• Simple example in a static system

analyte

gradient

Over time, analyte concentration at the surface will be depleted and a gradient will be generated through the liquid layer


Analyte consumption & supply

1. Analyte supplied by convection (continuous flow)

2. Diffusion becomes increasingly important as the flow rate reduces closer to the surface

3. Biomolecular interaction processes at the ligand/analyteinterface

flow cell height

diffusion

distance

1

2

3


Dealing with mass transport limitations

Low Rmax (ligand density)

High flow rates

• High flow rates reduce diffusion distance

Mass transport correction included in all kinetic models

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Experimental Design


Experimental designAffinity determination by steady-state analysis

Determine steady state binding levels over a range of analyte concentrations

High immobilization level

Concentration range should cover at least 20-80% saturation of the surface

Use reference surface

Include at least one concentration in duplicate

Include zero concentration sample


Important experimental parametersKinetic analysisThe purity of the reagents

Immobilization procedure

Immobilization level

Ligand activity

Flow rate

Analyte concentration range

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Analyte concentrationsKinetic analysis

Concentrations should cover a full range of binding curves

Include at least one concentration in duplicate

Include zero-concentration samples


1- What am I looking for?

1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Collect

information

about interacting

partners

• MW

• pI• stability

• solubility

• purity• activity



1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Choose suitable

immobilization

chemistry and

ligand density

Wait for a stable

baseline

Check for drifts

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1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Scout for buffer composition

• pH• ionic strength

• DMSO• metals• metal chelators

• BSA• cofactors

Keep it as simple as possible and match running buffer

and sample buffer



1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Check for a

positive control

• reproducibility• dose dependency• saturation

• stoichiometry

Check for a

reference control

• non specific binding• always check not subtracted

signals• run zero concentrations and

negative controls



1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Scout for

regeneration

• from mildest to harsh, never the reverse• try with regeneration

cocktails• always check for ligand activity and stability after

regeneration

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1. Specificity?

2. Ranking?

3. Kinetics?

4. Concentration?

5. Thermodynamics?

6. Other?

Adjust flow rate

• mass transport

limitation• rebinding

Adjust injection

parameters

• association phase• dissociation phase


Sensorgrams appearance

•Take time to look at the shapes of the sensorgrams

• Check for low quality data, such as drifts, air bubbles, spikes, aggregations

• Always have a look at what happens on the reference flow cell and on the active, not just at the subtracted

• Look at the curvatures of the sensorgrams if you are performing a kinetic analysis


Have a look...

Rich R and Myszka D, J. Mol. Recognit. 2008; 21: 355-400

A, B, C: Same kon but different koff

D: Equilibrium reached within seconds

E:Ligand saturation F: partial mass transport

limitation

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More complexity...

Is it a biphasic interaction?Maybe... But it is virtually impossible to resolve what actual event is leading to complex response

SOTake a step back and riconsider experimental design and conditions


Bad data sets

Substandard responses

• aggregation• precipitation• instrument maintenance

Drifting baseline

• ligand stability

• perform start up• regeneration issues


Data evaluation

What is the purpose of the

evaluation?

Yes/No data

Ranking

Equilibrium analysis

Kinetic rate analysis

Specificity studies

Early selectionof binders

Determination ofbinding strength

Modelling binding reactionsto determine the dynamic

behaviour of a system

KD

k , ka d

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Include zero-concentration samples

“Double referencing”

• Compensates for system disturbances

• Important for high-quality work

SampleReference-subtracted

Zero-conc

Double-referenced


Control experiments

Non-specific binding

Surface performance test

Regeneration optimization

Mass transport limitations

Linked reactions


Control experiments

Vary contact time at equilibrium to check for linked reactions

No linked reactions Linked reactions

Dissociation rates are not affected by contact time if reactionsare independent

Two-state reactions and competition are examples of linked reactions

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Summary

Affinity analysis

• Derives the affinity constants

• For analysis of interactions with very fast on and off rates

Kinetic analysis

• Derives the rate constants and the affinity constants

• For detailed characterization of a molecular interaction

• Interactions with the same affinity may have entirely different association and dissociation rate constants

• Rate constants may be more significant than affinity in

understanding biological processes

A

B

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Kinetic & Affinity Analysis - IQM CSIC · 1/ GE / Kinetic & Affinity Analysis An introduction 2/ GE Biacore T100 training_kinetics/ What are kinetics and affinity? Kinetics • How