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The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.

Rhodopseudomonas palustris uroporphyrinogen I synthetase (URO-S) has been chemically attached to Sepharose 4B and some of its properties have been studied. When 7-8 mg protein/ml activated Sepharose was used, immobilized URO-S retained 45% of the activity of the original soluble preparation, with a coupling yield of 66% after a period of 15 h. Optimal incubation conditions for the activity of gel-enzyme were determined. Unlike the soluble enzyme, the Sepharose-bound URO-S showed a biphasic substrate saturation curve, indicating that a protein conformational change had occurred during the process of immobilization. Immobilized URO-S stored at 4 degrees C for 35 days retained 90% of activity and when repeatedly used, up to 5 times, retained 48% of the original activity. Attachment of URO-S to Sepharose led to an enhanced thermal stability.