Laboratory Analysis of Enzymes and Isoenzymes – Diagnostic Enzymology

Flashcards cover measurement principles, Beer’s-Law-based calculations, manual vs automated kinetic methods, coupled reactions, immunoassays, isoenzyme analysis, and practical examples for Diagnostic Enzymology.

Under what kinetic condition should enzyme activity be measured for maximum accuracy?

When the reaction is at Vmax and follows zero-order kinetics.

Name the two fundamental ways to express enzyme activity under zero-order conditions.

1) Time needed to reach a fixed change in substrate concentration (inversely proportional to activity). 2) Amount of substrate converted (or product formed) in a fixed time (directly proportional to activity).

Which Beer’s Law variable is replaced by ∆A/min when calculating enzyme activity?

The absorbance term (A) is replaced by change in absorbance per minute (∆A/min).

Write the full equation (Step 4) for calculating enzyme activity (IU/L) from a kinetic assay.

IU/L = (∆A/min) × (Total reaction volume) / (ε × b × Sample volume).

What constant factor (F) is often pre-programmed into analyzers to simplify enzyme activity calculations?

F = (Total reaction volume)/(ε × b × Sample volume); enzyme activity is then ∆A/min × F.

Given ∆A/min = 0.100, total volume = 3.0 mL, sample = 0.2 mL, ε (NADH) = 6.33×10³ L mol⁻¹ cm⁻¹, and b = 1 cm, what is the enzyme activity?

237 IU/L (because 0.100 × 2370 = 237).

Why is NADH frequently chosen as the spectrophotometric indicator in coupled assays?

NADH absorbs strongly at 340 nm, whereas NAD⁺ does not, allowing clear monitoring of its concentration change.

In the AST assay, which reaction provides the chromophoric change measurable at 340 nm?

The indicator reaction: Oxaloacetate + NADH + H⁺ → L-Malate + NAD⁺ catalyzed by malate dehydrogenase (MD).

What is a coupled enzyme reaction?

A procedure in which a non-chromophoric primary reaction is linked to a secondary (indicator) reaction that produces or consumes a chromophore measurable by spectrophotometry.

List four general problems that can cause deviation from zero-order kinetics in an enzymatic assay.

Examples include substrate depletion, product inhibition, changes in pH or temperature, and enzyme instability or inhibitor presence.

On an absorbance-vs-time plot, which segment represents zero-order conditions?

The linear segment where absorbance changes at a constant rate (steady slope).

Describe the fixed-point method for kinetic measurements.

The reaction is allowed to proceed for a defined time, then the amount of product formed (or substrate left) is measured once.

Describe the continuous (multipoint) method for kinetic measurements.

Multiple absorbance readings are taken at fixed intervals while the reaction proceeds, allowing determination of reaction velocity in real time.

During a manual continuous assay, why must you identify the lag phase before collecting data?

Because enzyme-substrate complexes are still forming; data collected too early would not reflect steady zero-order velocity.

Which assay step is immediately followed by timed absorbance readings in the manual continuous method?

Completion of the lag phase.

What key calculation converts a three-minute ∆A/3 min value to the ∆A/min needed for activity determination?

Divide the ∆A/3 min by 3.

How do immunoassays differ from kinetic assays in enzyme analysis?

Immunoassays measure enzyme (or isoenzyme) protein mass, whereas kinetic assays measure catalytic activity.

Why can immunoassays detect inactive enzyme molecules while activity assays cannot?

Antibodies recognize antigenic determinants present on both active and inactive enzyme proteins.

Give two clinical situations where enzyme mass immunoassays are especially useful.

Isoenzyme or isoform quantitation, e.g., CK-MB determination; digestive enzymes like trypsin when inactive precursors or inhibitors are present.

Name three immunoassay formats used to measure enzymes or isoenzymes.

Direct, indirect, and sandwich (capture) immunoassays.

Which analytical techniques besides immunoassay are used to separate or quantify isoenzymes?

Isoelectric focusing, chromatography, chemical inactivation, and assays based on catalytic differences.

How does temperature influence the lag phase in an enzyme assay?

Higher temperatures shorten the lag phase by accelerating formation of the enzyme-substrate complex.

Why are no calibrators (standards) required when ε is known in kinetic enzyme assays?

Because molar absorptivity (ε) allows direct conversion of absorbance change to concentration change via Beer’s Law.

What role do controls play in enzyme assays even when standards are unnecessary?

Controls verify that reagents, instrumentation, and assay conditions yield expected results and remain within quality limits.

Define an International Unit (IU) of enzyme activity.

One micromole of substrate converted (or product formed) per minute under specified conditions per liter of sample.