3.1.5—Colorimetry in enzyme reactions

Colorimetry measures how much light a coloured sample absorbs or transmits. When the colour is linked to a substrate or product, the reading acts as a concentration proxy, allowing enzyme progress to be followed more objectively than judging colour by eye.

1. Set the reference: Use an appropriate blank to zero the colorimeter before measuring samples. Keep the cuvette clean and consistently oriented.
2. Calibrate the relationship: Prepare known concentrations, for example by serial dilution of a stock solution, and measure their absorbance or transmission. Plot concentration against reading to make a calibration graph.
3. Read unknowns: Measure the unknown reaction samples under the same conditions. Use the calibration relationship to estimate the corresponding substrate or product concentration.
4. Follow the rate: Take readings at defined times and plot the calibrated concentration, or a justified signal proxy, against time. The early linear gradient gives an initial rate; faster progress gives a steeper gradient.

A changing colour changes the amount of light absorbed or transmitted; calibration connects that reading to concentration; repeated timed readings then connect concentration change to enzyme rate. In a starch–amylase example, iodine colour decreases as starch is hydrolysed, so the calibrated decline in starch can be followed over time.

Absorbance or transmission is not automatically a concentration or a rate: the blank, standards, timing, cuvette handling and usable calibration range support that inference. This card does not assume a particular wavelength, unit or instrument specification.