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An acid phosphatase catalyses hydrolysis of phosphate esters under acidic conditions; its lysosomal enzyme latency supplied evidence for lysosomes.

Lysosomal enzyme latency is the difference between the enzyme activity accessible to an added substrate before disruption of the surrounding membrane and the activity revealed after disruption. Low accessible activity can reflect separation of enzyme from substrate, rather than absence of enzyme. This is an assay-accessibility statement, not a claim that the intact lysosome is inactive in the living biological cell.^[ref]

Let $F$ be the blank-corrected initial rate measured with a membrane-preserving preparation and $T>0$ the rate after a treatment that exposes all assayable enzyme. Use matched amounts of starting material, pH, substrate and measurement time. Define the apparent latent fraction byThis definition requires more than a large treated signal. Disruption must preserve enzyme activity, and the treatment must not independently alter the readout. A soluble-enzyme comparison with and without treatment helps test direct activation or inhibition; an appropriate blank tests signal unrelated to enzyme-catalysed hydrolysis. These are controls for interpreting the constructed assay, rather than additional historical observations.

For a deliberately chosen example, take $F=10$ and $T=100$ arbitrary rate units. Then $\ell =0.90$: ninety percent of the assayable activity was initially inaccessible. After hypothetical partial damage, $F=25$ with unchanged $T=100$ gives $\ell =0.75$. The preparation has lost latency without gaining total assayable activity. These percentages describe activity accessibility; they do not count intact lysosomes. Unequal enzyme contents, partial leakage and mixed particle populations prevent that identification. If $F>T$, the formula is negative, flagging failure of this simple accessibility interpretation rather than a negative population of hidden enzymes.

To check the logic, divide the same uniformly mixed preparation into two equal portions. If dilution halves both measured rates, $(F/2)/(T/2)=F/T$, so $\ell$ is unchanged. In contrast, comparing an undiluted untreated portion with a diluted treated portion changes the ratio artificially. Normalization to the same amount of original material is therefore part of the definition, not an optional numerical correction.

In the 1951 study by Jacques Berthet and Christian de Duve, liver from fasted rats was immediately chilled in an ice-cold isotonic chemical solution. The tissue was generally pressed through wire mesh; the weighed pulp was homogenized in cold $0.25\mathrm{mol}{\mathrm{L}}^{-1}$ sucrose using a glass Potter-Elvehjem homogenizer. Their acid phosphatase test concerned activity toward added glycerophosphate at pH $5$: the readout was substrate hydrolysis, rather than an image of a particle.^[ref]

Christian de Duve's retrospective describes successive centrifugation steps, separating centrifugation pellets and supernatants, and comparing enzyme distributions across the recovered fractions. Membrane disruption exposed previously inaccessible acid phosphatase activity, providing the crucial comparison with the membrane-preserving preparation. Joint fractionation patterns and release behavior of several acid hydrolases supported a shared population of digestive particles. By 1955, this evidence supported the lysosome concept.^[ref]

The inference was biochemical: a barrier restricted access to active enzymes. Co-sedimentation could confuse distinct particles; a crude mitochondrial fraction did not establish residence inside mitochondria. Lysosomal enzyme latency neither images an organelle nor determines its shape. Electron microscopy of enriched fractions supplied separate morphological evidence.^[ref]