ENZYMES AND METABOLIC NETWORKS

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AISHLEEN SINGH
A S S A Y S
E N Z Y M E

ENZYMES AND METABOLIC NETWORKS

 H E WELCOME TO
TODAY'S CLASS!
TODAY'S AGENDA
WELCOME TO TODAY'S CLASS / TODAY'S AGENDA

L
Introduction to enzyme assays 02

ENZYMES AND METABOLIC NETWORKS

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Types of Enzyme Assays 03

Enzyme assays based on sampling method 04

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Continuous Assays 06

Discontinuous Assays 08

The end 10

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 ENZYME ASSAYS
BRIEF INTRODUCTION
ENZYME ASSAYS / BRIEF INTRODUCTION

ENZYMES AND METABOLIC NETWORKS

The assay is the act of measuring how fast a given (unknown) amount of
enzyme will convert substrate to product (the act of measuring a velocity).
An assay requires to determine the concentration of a product or substrate at
a given time after starting the reaction.
Enzyme assays are laboratory methods for measuring enzymatic activity. They
are vital for the study of enzyme kinetics and enzyme inhibition.
Enzyme assays measure either the disappearance of substrate over time or the
appearance of product over time.

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 TYPES OF ASSAY
ENZYME ASSAYS / TYPES OF ASSAY

ENZYMES AND METABOLIC NETWORKS

INITIAL RATE PROGRESSIVE CURVE TRANSIENT KINETICS RELAXATION
EXPERIMENTS EXPERIMENTS EXPERIMENTS EXPERIMENTS

Rates are measured for a short
period after the attainment of
the quasi-steady state, typically
by monitoring the accumulation
of product with time.
The kinetic parameters are
determined from expressions for
the species concentrations as a
function of time.
reaction behaviour is tracked
during the initial fast transient as
the intermediate reaches the
steady-state kinetics period.
An equilibrium mixture of
enzyme, substrate and product is
perturbed, for instance by
a temperature, pressure or pH
jump, and the return to
equilibrium is monitored..

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 Continuous Assay
TYPES OF Where the assay gives a continuous reading
ENZYME ASSAYS / TYPES OF ASSAYS

ENZYMES AND METABOLIC NETWORKS

ENZYME of activity

ASSAYS
THEY CAN BE SPLIT
IN TWO CATEGORIES
ACCORDING TO Discontinuous Assay
THEIR SAMPLING Where the reaction is stopped to determine
METHOD
concentration

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 CONTINUOUS
ASSAY
ENZYME ASSAYS / CONTINUOUS ASSAYS

ENZYMES AND METABOLIC NETWORKS

where the assay gives a continuous reading of activity,
multiple measurements, usually of absorbance change, are made
during the reaction, either at specific time intervals (usually every 30
or 60 seconds)
or continuously by a continuous-recording spectrophotometer.  
These assays are advantageous over fixed-time methods because the
linearity of the reaction may be more adequately verified.

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 TYPES OF CONTINUOUS ASSAYS
ENZYME ASSAYS / TYPES OF CONTINUOUS ASSAYS

ENZYMES AND METABOLIC NETWORKS

Spectrophotometry Fluorescence Calorimetry

Chemiluminiscence Light Scattering Microscale Thermophoresis

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 DISCONTINUOUS
ASSAYS
ENZYME ASSAYS / DICONTINUOUS ASSAYS

ENZYMES AND METABOLIC NETWORKS

Discontinuous assays, where samples are taken, the reaction stopped and
then the concentration of substrates/products determined.
the reaction proceeds for a designated time,  
the reaction is stopped (usually by inactivating the enzyme with a
weak acid),
 a measurement is made of the amount of reaction that has occurred.

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 ENZYME ASSAYS / TYPES OF DISCONTINUOUS ASSAYS

RADIOMETRY

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TYPES OF DISCONTINUOUS ASSAYS

CHROMATOGRAPHY

ENZYMES AND METABOLIC NETWORKS

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ENZYMES AND METABOLIC NETWORKS