A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.

A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The Mr of the native protein, determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities. Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4.4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A405/A280 ratio never exceeded 0.27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the gamma, beta, and alpha bands were at 441, 559 and 590 nm respectively, the alpha-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent gamma-band at 426 nm and caused significant shifts of the alpha and beta bands to shorter (574 and 545 nm) wavelengths. The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: Vmax was 2.3 X 10(6) mol H2O2 (mol haem)-1 min-1 and the Km was 11 mM. At 10 mM-H2O2 the first order rate constant was 0.3 X 10(7) M-1 s-1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3',6-trichloroindophenol as substrates; for the latter substrate, the Km was 0.18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.