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Enzymatic purity is the fraction of the activity observed in a particular enzyme assay attributable to the intended enzyme. It concerns the origin of the measured reaction signal. A preparation can have low protein mass purity but high enzymatic purity if the other proteins contribute no activity in that assay.^[ref]

A constructed counterexample shows why mass purity is insufficient. Suppose a mixture contains $99\mu \mathrm{g}$ target and $1\mu \mathrm{g}$ contaminating enzyme. In the chosen assay, let their activities be 1 and 100 units per microgram respectively. The two contributions are 99 and 100 units. Target mass purity is $99\%$, but its fraction of the signal is $99/199\approx 49.7\%$.

Separating those contributions needs experimental evidence. Comparing an active target with an otherwise matched inactive-target preparation can reveal activity surviving without target catalysis. Substrate choices, inhibitor responses and independently purified preparations provide complementary tests.^[ref]

The conclusion remains assay-specific: a contaminant may be silent for one substrate yet active for another. Specific activity reports total activity per protein mass; it does not by itself identify which enzyme produced that activity.

John E. Scott is an enzyme assay researcher and coauthor, with Kevin P. Williams, of guidance on validating enzyme preparations. The original byline associates both authors with the BRITE institute at North Carolina Central University when the chapter was prepared.^[ref]

Their methodological contribution separates three questions: is the intended enzyme present, what fraction of the protein material does it represent, and what fraction of the measured activity actually comes from it? Protein mass purity and enzymatic purity are different because a small contaminant can contribute disproportionately to an enzyme assay.^[ref]

Consider an invented preparation containing $99$ mass units of the intended enzyme and one of a contaminant. If that contaminant produces $100$ times as much assay signal per unit mass, the two components contribute $99$ and $100$ signal units. The intended enzyme supplies only $99/199\approx 49.7\%$ of the measured signal despite $99\%$ protein mass purity.

Kevin P. Williams is a researcher in enzyme assay methodology and coauthor, with John E. Scott, of guidance on assessing enzyme preparations. Their chapter distinguishes enzymatic purity from both protein mass purity and specific activity.^[ref]

The practical contribution is a strategy for testing where an assay signal comes from. Inhibitor-based comparisons, changes of substrate and comparison of assay formats can expose unwanted activities that a clean-looking SDS-PAGE band would miss.^[ref]

For example, suppose a control selectively suppresses the intended enzyme but a substantial signal remains. That observation motivates checking for another signal source; it does not, by itself, identify a second enzyme. Pairing that control with an independent enzyme assay or a different substrate helps distinguish biochemical contamination from a measurement artefact.

Protein mass purity is the fraction of the total protein mass in a preparation contributed by the intended protein. With target mass $m_T$ and total protein mass $m_P$, the percentage is $100m_T/m_P$. The denominator includes other proteins, rather than the solvent or the entire mass of the solution.^[ref]

For a constructed preparation containing $0.84\mathrm{mg}$ target protein and $0.16\mathrm{mg}$ other proteins, mass purity is $84\%$. This does not say that $84\%$ of the target molecules are active, nor that $84\%$ of measured catalytic activity comes from them. Those are different quantities from a mass fraction.

An image-based estimate also requires a response model. Suppose densitometry gives target-band intensity 8 and contaminant intensity 2. If both stain equally per unit mass, the inferred purity is $8/(8+2)=80\%$. If, instead, the target signal per mass is twice the contaminant's, the inferred masses are proportional to 4 and 2, giving $4/6\approx 67\%$. These invented response factors illustrate the assumption rather than a measured staining difference.

Thus an SDS-PAGE band pattern supports a mass-purity estimate when identification, detection and calibration are appropriate. Enzymatic purity and specific activity require additional assay information.