 Enzyme inhibition initial-rate assay

The protocol used in the trypsin colorimetric assay requires $40$–$60\%$ inhibition and rejects hazy or yellow BAPNA solution.^[ref]

The trypsin colorimetric assay explains blank subtraction and conversion to a reaction rate using the Beer-Lambert law. This protocol is an activity comparison at one substrate level, not evidence that aprotinin obeys any particular curve above. To test the model, an experimental extension would vary both free inhibitor and free substrate concentrations, estimate early linear reaction rates before appreciable depletion, and fit the whole collection by nonlinear regression. Include a no-inhibitor series and inhibitor-matched blanks: inhibitor-dependent absorbance drift must not be mistaken for changed catalysis.

There is a mathematical reason to collect more than one reaction rate. At $s=20\mu \mathrm{M}$ with the chosen uninhibited parameters, measuring $v=25\mu \mathrm{M}\mathrm{mi}{\mathrm{n}}^{-1}$ would imply only $\alpha +{\alpha }^{\prime }=4$. Both $(\alpha ,{\alpha }^{\prime })=(2,2)$ and $(3,1)$ satisfy that observation, although their high-$s$ limits differ. A broader substrate range tests which model fits. Even a fitted curve leaves binding geometry unresolved. Check activity recovery after reducing inhibitor concentration separately; the simple equation also ceases to apply when free inhibitor is substantially depleted by binding or inhibitor exchange has not equilibrated during the measured interval.