Enzyme Activity Calculator

A continuous spectrophotometric assay converts a slope into a rate. Beer's law states that absorbance equals the molar extinction coefficient times path length times concentration, so dividing the absorbance change per minute by the product of epsilon and path length gives the molar rate of appearance or disappearance of the chromophore. Multiplying by reaction volume converts concentration change into an amount, and one international unit is defined as one micromole of substrate transformed per minute. Dividing total units by the volume of enzyme added gives volumetric activity in U/mL; dividing instead by the milligrams of protein added gives specific activity in U/mg, the number that tracks purification. Common coefficients are 6220 per molar per cm for NADH at 340 nm, 18300 for p-nitrophenolate at 405 nm and 14150 for the TNB anion released from Ellman reagent at 412 nm. The measurement is only meaningful as an initial velocity, which in practice means restricting the reading to the linear region where less than about 10 percent of substrate has been consumed; beyond that, substrate depletion and product inhibition bend the curve and the rate is underestimated. Extinction coefficients are condition dependent, since the p-nitrophenolate value assumes alkaline pH and the phenol has a pKa near 7.1, and microplate wells do not have a 1 cm path, so a 200 microlitre well is closer to 0.55 cm unless the instrument applies path length correction. Activity is defined only against a stated temperature, pH and substrate concentration.

Quantitative enzymology needed a common currency. Leonor Michaelis and Maud Menten published their treatment of invertase kinetics in 1913, giving the field a rate equation, but laboratories still reported activity in idiosyncratic house units well into the 1950s, which made results from different groups impossible to compare. The Commission on Enzymes of the International Union of Biochemistry addressed this in its 1961 report, which both introduced the EC numbering scheme for enzyme classification and defined the enzyme unit as the amount catalysing transformation of one micromole of substrate per minute under stated conditions. The SI-consistent replacement, the katal, defined as one mole per second, was recommended by IUB and IUPAC in the early 1970s and formally accepted as a derived SI unit by the General Conference on Weights and Measures in 1999; one unit equals about 16.67 nanokatals. Clinical chemistry adopted katals in some countries while biochemistry largely kept the unit. The optical tools arrived alongside: the absorption law traces to Pierre Bouguer and Johann Heinrich Lambert in the eighteenth century and August Beer in 1852, Arnold Beckman's DU spectrophotometer of 1941 put reliable ultraviolet readings on the bench, Horecker and Kornberg established the NADH coefficient at 340 nm in 1948, and George Ellman described his thiol reagent in 1959.