A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.

Chymopapain (EC 3.4.22.6) was purified from commercially available dried latex of papaya (Carica papaya) by extraction at acidic pH, cation-exchange chromatography and active site-directed affinity chromatography on immobilized alanyl-phenylalaninaldehyde semicarbazone, with elution by mercuric chloride. The product was found by immunoassay to be essentially free of the other cysteine proteinases from papaya, including papaya proteinase IV, and was fully active.The rate of alkylation of the active site cysteine of Chymopapain by iodoacetate was found to be sufficiently rapid and selective for this reagent to be used as an active-site titrant. Präparation von voll aktiven Chymopapain, frei von kontaminierenden ProteinasenZusammenfassung: Chymopapain (EC 3.4.22.6) wurde aus handelsüblicher getrockneter Papaya-Latex (Carica papaya) durch Extraktion bei saurem pH, Kationen-Austausch-und Affinitätschromatographie über die Bindung des Aktivitätszentrums an immobilisiertes Alanylphenylalaninaldehyd-semicarbazon und Elution durch Quecksilberchlorid gereinigt. Das Produkt war vollständig aktiv und, wie durch Immunoassay belegt, frei von anderen Papaya-Cysteinproteinasen einschließlich Papaya-Proteinase IV. Die Alkylierung des Cysteins im aktiven Zentrum von Chymopapain mit lodessigsäure verlief hinreichend schnell und selektiv, so daß dieses Reagenz zur Titration des aktiven Zentrums benutzt werden kann.

A comparison of the product-inhibition patterns during cleavage of D-fructose 1,6-diphosphate by aldolases from yeast, rabbit muscle and Bacillus stearothermophilus shows an ordered reaction sequence for all three enzymes, with dihydroxyacetone phosphate the last-leaving product. Addition of Zn2+, Co2+, Fe2+, Mn2+ or Cd2+ ions to the inactive apo-(Bacillus stearothermophilus aldolase) restores activity to different extents, whereas Ni2+, Mg2+ or Cu2+ ions have no effect. The cleavage activity of this aldolase is not enhanced by added K+ ion. The effects of metal replacement on thermal stability, Km and Vmax. are given and the possible role of the metal is discussed in the light of these results.

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