= Enzyme inhibition initial-rate assay
The protocol used in the <trypsin colorimetric assay> requires $40$–$60\%$ inhibition and rejects hazy or yellow <BAPNA> solution.<Enzymatic Assay of APROTININ - Sigma Quality Control Test Procedure>[A6012, SPBAPN02.001, revised 22 December 1997, p. 3, Notes 1–2: target inhibition range and substrate-solution quality]{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 $s=20\,\mathrm{\mu M}$ with the chosen uninhibited parameters, measuring $v=25\,\mathrm{\mu M\,min^{-1}}$ would imply only $\alpha+\alpha'=4$. Both $(\alpha,\alpha')=(2,2)$ 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.
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