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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.

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.

Protein purification separates a selected protein from other components of a sample while recovering material useful for a subsequent measurement or application. Different procedures exploit different properties. In affinity chromatography, a matrix-bound ligand selectively retains the target through a reversible interaction.^[ref]

An enzyme example separates recovery from enrichment. Suppose a starting fraction contains $10\mathrm{mg}$ total protein and $200$ measured activity units, while a collected fraction contains $2\mathrm{mg}$ and $120$ units. The specific activity rises from $200/10=20$ to $120/2=60$ units per milligram: a threefold increase. Recovery of activity is $120/200=0.60$, or $60\%$. These are constructed assay values, not reported experimental data.

The preparation can therefore be enriched even while some target activity is lost. That inference assumes the assay reports the same catalytic property under comparable conditions; activation or inhibition can change activity without an equivalent change in protein amount. Protein purification consequently needs a stated target, an identity or activity measurement, and a recovery criterion. A visually clean fraction alone does not establish that its protein remains functional.

Specific activity is measured enzymatic activity divided by the total protein mass in the preparation. It describes activity per mass, commonly reported in assay units per milligram, and is distinct from total activity or the fraction of the preparation consisting of the intended enzyme.^[ref]

For a constructed protein purification example, a fraction with $240$ activity units and $12\mathrm{mg}$ total protein has specific activity $20\mathrm{U}\mathrm{m}{\mathrm{g}}^{-1}$. A later fraction with $150$ units and $3\mathrm{mg}$ gives $50\mathrm{U}\mathrm{m}{\mathrm{g}}^{-1}$: enrichment is $50/20=2.5$-fold, while activity recovery is $150/240=62.5\%$. The denominator includes other proteins, not just the target.

The comparison requires compatible enzyme assay conditions. A higher value can reflect removal of inactive material, but can also follow activation or removal of an inhibitor. Moreover, a contaminating enzyme may perform the measured reaction. High specific activity therefore supports an assessment of a preparation without independently establishing its molecular identity or proving that all measured activity comes from the desired catalyst.^[ref]