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Lecture 15

BIOL 200 Lecture Notes - Lecture 15: Protein Structure, Calorimeter, Fluorescent Tag


Department
Biology (Sci)
Course Code
BIOL 200
Professor
Mathieu Roy
Lecture
15

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Even when the enzyme reaction does not result in a change in the absorbance of light, it can still
be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the
product of one reaction is used as the substrate of another, easily detectable reaction. For
example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by
coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-
phosphate dehydrogenase.
Fluorescence is when a molecule emits light of one wavelength after absorbing light of a
different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from
product to measure the enzyme reaction. These assays are in general much more sensitive than
spectrophotometric assays, but can suffer from interference caused by impurities and the
instability of many fluorescent compounds when exposed to light.An example of these assays is
again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are
fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be
followed by a decrease in fluorescence and reduction reactions by an increase. Synthetic
substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such
as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.
it is the measurement of the heat released or absorbed by chemical reactions. These assays are
very general, since many reactions involve some change in heat and with use of a
microcalorimeter, not much enzyme or substrate is required. These assays can be used to
measure reactions that are impossible to assay in any other way.
Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions
produce light and this can be measured to detect product formation. These types of assay can be
extremely sensitive, since the light produced can be captured by photographic film over days or
weeks, but can be hard to quantify, because not all the light released by a reaction will be
detected.The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a
common method of detecting antibodies in western blotting. Another example is the
enzyme luciferase, this is found in fireflies and naturally produces light from its substrate
luciferin.
Static light scattering measures the product of weight-averaged molar mass and concentration of
macromolecules in solution. Given a fixed total concentration of one or more species over the
measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of
the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies
the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics
is a very general technique that does not require an enzyme.
Microscale Thermophoresis (MST measures the size, charge and hydration entropy of
molecules/substrates in real time. The thermophoretic movement of a fluorescently labeled
substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be
measured with high time resolution in real time. The material consumption of the all optical
MST method is very low, only 5 µl sample volume and 10nM enzyme concentration are needed
to measure the enzymatic rate constants for activity and inhibition. MST allows to measure the
modification of two different substrates at once (multiplexing) if both substrates are labeled with
different fluorophores. Thus substrate competition experiments can be performed.
Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the
amount of product production or substrate consumption is measured in these samples.
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