Enzyme Assays and Automation – Part I

Question-and-answer flashcards covering purposes, conditions, and multiple detection methods (manometry, enthalpimetry, spectrofluorimetry, bioluminescence, electrochemical, radiochemical, and solid-phase) used in enzyme activity assays.

List three major reasons scientists perform enzyme assays.

1) To employ enzymes as biotechnology tool kits (synthesis or analysis), 2) To understand disease mechanisms and severity, 3) To develop or evaluate enzymes as drug targets or drugs themselves.

What two clinical uses arise from measuring enzyme activity?

Disease diagnosis (determining what is wrong) and disease monitoring (gauging severity or progression).

Which five reaction conditions must be optimized before running an enzyme assay?

Optimal pH, ionic strength, temperature, absence of inhibitors, and availability of necessary cofactors or coenzymes.

Why is substrate concentration adjusted to around the KM value during assays?

Assaying near KM allows accurate measurement of velocity changes that are sensitive to enzyme activity without saturating the enzyme.

Name four common detection techniques for measuring enzyme activity.

Spectrophotometry, spectrofluorimetry, calorimetry/enthalpimetry, manometry, plus electrochemical, radiochemical, and dry-reagent methods.

In manometric assays, which physical parameter is directly monitored?

The change in pressure (or gas volume) produced or consumed by the enzymatic reaction.

Give one example of an enzyme routinely measured by manometry.

Glucose oxidase (oxygen consumption) or a decarboxylase (CO₂ production).

State two advantages of enthalpimetry for enzyme assays.

High sensitivity and freedom from many interfering substances; it can be adapted to diverse reactions.

What physical quantity does enthalpimetry measure during an enzyme reaction?

The enthalpy (heat) change generated or absorbed by the reaction.

Hexokinase shows an enthalpy change (ΔH) of approximately in enthalpimetric assays.

−28 kJ mol⁻¹.

How does spectrofluorimetry detect enzyme activity?

By attaching or generating a fluorophore whose fluorescence intensity changes proportionally with product formation or substrate depletion.

Write the fluorescence intensity relationship used at low concentrations in spectrofluorimetric assays.

If − Io = 2.3 E c l q, where E is molar absorptivity, c concentration, l path length, q quantum efficiency.

Give a substrate–product example that illustrates a fluorogenic lipase assay.

Dibutyryl fluorescein (non-fluorescent) → Fluorescein (fluorescent) via lipase action.

What clinical problem was addressed using a fluorescent enzyme assay published in Chem. Sci., 2017?

Assessment of acute pancreatitis.

What intracellular enzyme does the bioluminescent ADCC assay from BioTek measure upon cell lysis?

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Why does the BioTek ADCC assay avoid pre-labelling or transfection steps?

Because it detects naturally released GAPDH, eliminating the need to introduce artificial reporter molecules.

Differentiate potentiometric techniques from voltammetry in electrochemical assays.

Potentiometric methods measure the potential generated by the reaction at zero current, whereas voltammetry (polarography) applies varying voltage and measures the resulting current changes.

Which section of a voltammogram is diagnostic for analyte concentration in enzyme-coupled assays?

The current plateau or peak corresponding to the analyte’s redox potential.

Name four radioisotopes frequently used in radiochemical enzyme assays.

³H (tritium), ³²P, ³⁵S, and ¹³¹I.

After an enzymatic reaction with a radiolabelled substrate, how are substrate and product usually separated?

By electrophoresis or chromatography, followed by quantifying the radioactive fraction.

List two major advantages of radiochemical enzyme assays.

Very high sensitivity (picomolar range) and the ability to track specific atoms through complex reaction mixtures.

What is one practical benefit of immobilising enzymes or substrates on a solid phase for ‘dry-reagent’ assays?

It enables portable, point-of-care kits that are easy to use and can provide fully quantitative or semi-quantitative results without liquid reagents.