Water-insoluble enzymes: Arrangement of aldolase within an insoluble carrier

Crystalline, radioactive muscle aldolase was prepared from a rabbit injected subcutaneously with a mixture of fifteen 14C-labeled amino acids. The radioactive enzyme exhibiting 1.4 × 10 −4 μC per mg protein was transformed into a water-insoluble state by embedding it into a highly cross-linked, insoluble synthetic polyacrylamide carrier, according to the procedure previously described by us ( Science 142: 678, 1963). The distribution of enzyme activity and of total enzyme protein, as measured by the radioactivity, between insoluble carrier and remaining aqueous phase was determined. While only 44.5% of the total enzymic activity could be recovered, all of the radioactivity was accounted for. The ratio of enzyme activity to radioactivity in the aqueous phase, which remained after termination of the polymerization, was not much lower than the corresponding ratio of the original soluble aldolase (25% reduction). This suggests that the contact of enzyme with the reagents for polymerization caused no more than 25% enzyme inactivation. However, the respective ratio in the insoluble phase was four times smaller, indicating that four times more enzyme protein than enzymic activity was associated with the carrier. The most plausible explanation of these findings is the assumption that only that portion of the embedded aldolase deploys enzymic activity, which is located at or near the surface of the carrier particle. Once the aldolase is embedded in the synthetic polymer, the enzyme can no longer be separated from its insoluble carrier.