This document discusses various instrumental techniques that can be used for enzymatic analysis, including manometry, spectrophotometry, spectrofluorimetry, electrochemical methods, enthalpimetry, radiochemical methods, and dry-reagent techniques. It also covers automation in enzymatic analysis and high-throughput assays. Some key techniques mentioned are manometry to monitor reactions involving gases, spectrophotometry using the Beer-Lambert law to follow reactions involving NADH/NADPH, and radiochemical methods using radioactively labeled substrates to measure product formation over time. Fully automated procedures allow simultaneous analysis of multiple samples without manual intervention between steps.

1. INSTRUMENTAL TECHNIQUES
AVAILABLE FOR USE IN ENZYMATIC
ANALYSIS
By
Perwez Bakht

2. CONTENTS
• Detection techniques
• Introduction
• Manometry
• Spectrophotometry
• Spectrofluorimetry
• Electrochemical methods
• Enthalpimetry
• Radiochemical methods
• Dry-reagent techniques
• Automation in enzymatic analysis
• High-throughput assays (HTA)

3. introduction
• A variety of detection procedures are used, or have been used in the past, for
enzymatic analysis by either end-point or kinetic methods.
• But in most cases, the instrument detecting the changes which take place as the
reaction proceeds can be coupled to a recorder and/or computer, enabling the
course of the reaction to be followed in detail without requiring constant human
attention.
• Also, the enzyme involved may be in an immobilized form to allow it to be
recovered at the end of the procedure.

4. manometer
• If any of the substrates or products of an enzyme-catalysed reaction is a gas, the
course of the reaction may be followed by manometric techniques.
• The sample and reagents are put into different compartments in the reaction
vessel and mixed together at zero time; then, as the reaction proceeds, the
uptake or evolution of gas is indicated by the movement of fluid in the
manometer tube.
Manometric methods may be used to
investigate reactions where a gas is
consumed, e.g. that catalysed by glucose
oxidase, where molecular oxygen is a
substrate.
They may also be used when a gas is
produced, e.g. to monitor C02
production in reactions catalysed by
decarboxylases

5. spectrophotometer
• If a substrate or a product absorbs light at a characteristic wavelength, then the
reaction can be monitored by following the changes in absorbance at this
wavelength.
• Spectrophotometric methods are particularly useful for monitoring reactions
which involve NAD+ or NADP+ as coenzyme , or ones which can be coupled to
such reactions.
• Beer-lambert law:-

6. spectrofluorimeter
• Compounds are said to be fluorescent when they absorb light of one wavelength
and then emit light of a longer wavelength.
• At low concentrations, the intensity of fluorescence (fr) is related to the intensity
of the incident light (fo) of appropriate wavelength by the relationship:
• where is the molar absorption coefficient, c the molar concentration, l the
length of the light-path and q the quantum efficiency (i.e. the number of quanta
fluoresced divided by the number of quanta absorbed)
• NADH and NADPH exhibit fluorescence, absorbing light at 340 nm and reemitting
it at about 460 nm. Therefore, any reactions utilizing these as coenzymes may be
followed by the use of sensitive spectrofluorimetric procedures.
• Other example:-

7. electrochemical methods
• Many different types of electrochemical procedures have been used in enzymatic
analysis. Often these involve potentiometric techniques, where an electrical
potential is generated which is dependent on the concentration and properties of
substances in the test solution; the change in potential as the reaction proceeds
can be taken to be a measure of the rate of the reaction.
• Other types of electrochemical procedure include those based on polarography
or voltammetry, where an increasing voltage is applied between two electrodes
immersed in the test solution, whose composition determines the current which
flows at each instant.
• For those enzyme-catalysed reactions where there is a change in the number of
charged species as substrates are converted to products, the course of the
reaction may be followed by determining the change of electrical conductance
with time: this is termed conductometry.

8. enthalpimetry
• Enzymatic analysis by enthalpimetry (microcalorimetry) is of potential
importance because of the sensitivity, freedom from interference and almost
universal possibilities of application of these techniques.
• Extremely accurate thermostatting (temperature range- ) or
excellent insulation is required, together with very sensitive temperature
sensors.
David McGlothlin and Joseph Jordan (1975)
used enthalpimetry to estimate blood
glucose by means of a hexokinase-catalysed
reaction in Tris buffer at pH 8:

9. radiochemical methods
• The use of a radioactively-labelled substrate can be valuable in enzymatic
analysis. The isotopes most commonly used for labelling purposes are 3H
(tritium), 14C(carbon), 32P(phosphorous), 35S(sulphur) and 131I(iodine). All of
these isotopes emit beta-radiation (electrons) as they decay.
• After the enzyme-catalysed reaction has progressed for a specified period, it is
terminated. The substrate is then separated from the product, usually by
chromatography or electrophoresis, and the product concentration is
determined indirectly by measuring the radioactivity of the product fraction.
• A typical example of enzymatic analysis by a radiochemical procedure is that
involving the cholinesterase-catalysed hydrolysis of [ 14C]-acetylcholine:

10. dry-reagent techniques
• For a number of years, test strips have been available for certain routine
analytical procedures.
• The strip, now usually made of porous plastic, incorporates a pad at one end
impregnated with the reagents, which may include enzymes. This end of the strip
is dipped into the test solution, and, after a specified time, the colour produced
in the pad is compared with those on the chart provided.
• A semi-quantitative assessment of the concentration of a reactant in the test
solution is thus obtained.
• Such a procedure is widely used to give an indication of the D-glucose content of
urine .

11. AUTOMATION IN ENZYMATIC ANALYSIS
• Automation or automatic control, is the use of various control systems for
operating equipment such as machinery, processes in factories, boilers and heat
treating ovens, switching in telephone networks, steering and stabilization of
ships, aircraft and other applications with minimal or reduced human
intervention.
• As discuss above, most of the detectors used to monitor the course of enzyme-
catalysed reactions can be coupled to automatic recorders. However, if each
sample still has to be mixed with reagents and introduced to the detector by
hand, the overall process can only be said to be semi-automatic.
• Fully-automatic procedures, where simultaneous or sequential analysis can be
carried out on a batch of samples, without any manual steps being involved
other than in sample preparation and in loading the batch on to the analyzer,
and possibly in the calculation of the results from recorder traces.

12. HIGH-THROUGHPUT ASSAYS (HTA)
• To screen the large and increasing number of available chemicals,
that may form the basis of drug discovery, robotics are required to
simultaneously test the compounds in functional or binding assays, a
process called high throughput screening (HTS).
• Multi-well plastic plates (typically 96, 384 or 1536 wells) provide the
means to interact small volumes (typically :<10µl) of sample (e.g.
enzyme or cells) with stock solutions of the compound library.
• Chromogenic, fluorescent, luminescent and radiochemical
techniques can be used as a means of detecting the occurrence of
enzyme inhibition.