Spectroscopy, Enzyme Assays & Quantitative Analysis – Review Flashcards

These flashcards review key wavelengths, enzyme functions, assay protocols, calibration-curve math, dilution calculations, carbohydrate basics, and best practices for UV–Vis and fluorescence assays.

What wavelength range is considered the ultraviolet (UV) region in UV–Vis spectroscopy?

~200–400 nm

What wavelength range is considered the visible region in UV–Vis spectroscopy?

~400–800 nm

In an absorbance assay, the instrument measures a ___ in transmitted light at the selected wavelength.

decrease

Why is fluorescence detection generally more sensitive than absorbance detection?

Only fluorescent compounds emit light, so background is low, giving higher selectivity and sensitivity.

At what wavelength is NADH monitored in the hexokinase glucose assay?

Absorbance at 340 nm

The Fiske–Subbarow phosphate assay measures absorbance at approximately what wavelength?

~600 nm

In the fluorescent protease assay, what are the excitation and emission wavelengths?

Excitation 485 nm; Emission 535 nm

Which two enzymes are used in the coupled glucose assay?

Hexokinase and glucose-6-phosphate dehydrogenase (G6PDH)

What product of the glucose assay is actually detected spectroscopically?

NADH, formed by G6PDH and absorbing at 340 nm

What is the purpose of generic phosphatase in the phosphatase assay?

To hydrolyze phosphate groups from biomolecules, releasing inorganic phosphate (Pi).

Which reagent system (no enzyme) detects inorganic phosphate colorimetrically?

The Fiske–Subbarow reagent (acidic molybdate + reducing agent).

What enzyme in the fluorescent protease assay releases fluorescein from FITC-casein?

Trypsin (a serine protease).

Do enzymes change reaction equilibrium or reaction rate?

They change the rate only; equilibrium position remains unchanged.

In a calibration curve, which variable is plotted on the x-axis?

Known concentration (or activity) of the standard.

Write the linear regression equation used to find unknown concentration from a calibration curve.

y = m x + b, so x = (y – b) / m

What statistical value indicates the goodness-of-fit for a linear regression?

R² (coefficient of determination)

State the dilution formula for preparing a working solution from a stock.

Cdil = (Vstock × Cstock) / (Vstock + V_solvent)

Convert 1 mM to micromolar (µM).

1 mM = 1,000 µM

Convert 1 mg/mL to µg/mL.

1 mg/mL = 1,000 µg/mL

Name the three common monosaccharides listed in the notes.

Glucose, fructose, and galactose

Which enzyme converts glucose-6-phosphate to fructose-6-phosphate?

Phosphoglucose isomerase

What two key features make fluorescence instrumentation more complex than absorption instruments?

Separate excitation and emission filters (or monochromators).

Which detection mode (absorbance or fluorescence) generally offers higher sensitivity?

Fluorescence

According to best practice, how should standards and unknowns be run in microplate assays?

Side-by-side in triplicate

What minimum R² value usually indicates good linearity for a standard curve in these assays?

R² ≥ 0.98

Why is it important to include error bars (±SD) on calibration plots?

They show assay precision and help evaluate variability among replicates.