J. Biochem, 1976, Vol. 79, No. 6 1223-1234
© 1976 Japanese Biochemical Society
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Purification and Characterization of a Proteinase from Pineapple Fruit, Fruit Bromelain FA21
Department of Biochemistry, School of Medicine, Nagoya City University Mizuho-ku, Nagoya, Aichi 467
Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized.
1.Efficient extraction of this enzyme from the crude material was possible using " Cellulosin AP, " a microbial polysaccharidase preparation containing cellulase, hemi-cellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31, 000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2.
2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein.
3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined.
Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS
4. FA2 showed pH optima of 8.0 and 8.3 toward hemoglobin and casein, respectively. The Michaelis constant for 
-N-benzoyl-L-arginine ethyl ester was 43 mM at pH 6.0 and 25°, FA2 cleaved bradykinin between Gly4-Phe5 and between Phe5-Ser6 at comparable rates; it hydrolyzed angiotensin II preferentially between Tyr4-lle5.
5. Similarities and differences found thus far between fruit bromelain FA2, stem bromelain SB1, and papain are tabulated.
1This work was supported in part by a grant from the Ministry of Education, Science and Culture, of Japan. A part of this work is taken from a dissertation submitted by F. Yamada to the Graduate School of Nagoya City University in partial fulfillment of the requirements for the degree of Doctor of Medical Science, Feb. 1974.
2Present address: T. Murachi, Department of Clinical Science, Kyoto University Hospital, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto, Kyoto 606.
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