Papain, Chymo

I.U.B.: 3.4.22.6

Papain is a sulfhydryl protease from Carica papaya latex. (A second protease, chymopapain, and a lysozyme have also been isolated from this same source.) Since native crystalline papain is quite unreactive until acted upon by mild reducing agents such as cysteine or cyanide, it may exist as a zymogen (Brocklehurst and Kierstan 1973). For a general review, see Liener (1974). Barrett and Buttle (1985), Polgár (1984) and Brocklehurst and Salih (1983) report on the classification of papaya latex proteinases. Papain has wide specificity. In her review, Arnon (1970) has indicated that it will degrade most protein substrates more extensively than the pancreatic proteases. It is also an esterase. Papain has been reviewed by Smith and Kimmel (1960). It has been reported by Sluyterman and Wijdenes (1972) that the action of papain on leucine methyl ester produces an insoluble polyleucine peptide. The finding of Thomas (1956) that papain breaks down the intercellular matrix of cartilage (see also McCluskey and Thomas 1958), led to its further study as a chondromucoproteinase (Smith et al. 1962).

Proteolytic enzymes are widely used in cell isolation. With some tissues papain has proved less damaging and more effective than other proteases. Lam (1972) found that of the enzymes used for dissociating turtle retina, papain produced the least trauma. Intact single photoreceptor cells have also been isolated from adult salamander retina with papain (Bader et al. 1978, Townes-Anderson et al. 1985). Huettner and Baughman (1986) descrbed a method using papain to obtain high yields of viable, morphologically intact cortical neurons from postnatal rats. Finkbeiner and Stevens (1988) applied the Huettner and Baughman method to the dissociation of postnatal rat hippocampus. Papain is used with fetal as well as postnatal brain regions to provide maximal dissociation and viability of neurons.

Characteristics of Papain from Carica Papaya:

Molecular weight: 23,000 (Dreuth et al. 1968).

Composition: Papain is a single peptide chain of 211 residues folded into two parts that form a cleft (Dreuth et al. 1968). A three-dimensional structure has been indicated by Wolthers et al. (1970). The molecule has one free SH group which is functional (Smith et al. 1975; Shipton et al. 1975). According to Alecio et al. (1974) there are seven subsites each capable of accommodating a single amino acid residue of a peptide substrate. See also Glick and Brubacker (1974). Other reports on molecular information and its relation to activity are as follows: Fink and Gwyn (1974), Lewis and Shafer (1974), Akalski et al. (1973), Allen and Lowe (1973), Brocklehurst and Little (1973), Mole and Horton (1973), Banks and Shafer (1972), Brocklehurst et al. (1972), Campbell and Kaiser (1971, 1972), Sluyterman and Wijdenes (1972), Hinkle and Kirsch (1971) Jori et al. (1971), Lowe and Yuthavong (1971) and Steiner (1971).

Optimum pH: 6.0 - 7.0.

Extinction coefficient: = 25.0 (Mitchel et al. 1970).

Isoelectric point: pH 9.6 (Sluyterman and DeGraff 1972).

Activators: Papain is activated by cysteine, sulfide, sulfite, etc. It is enhanced when heavy metal binding agents such as EDTA are also present. Kirschenbaum (1971) indicated that N-bromosuccinimide enhances the activity. Hall et al. (1972) report on the affects of acridine dyes.

Inhibitors: Substances which react with sulfhydryl groups including heavy metals, carbonyl reagents (Morihara 1967). Westerik and Wolfenden (1972) have studied aldehydes as papain inhibitors and Sluyterman and Wijdenes (1973) report on benzoylamidoacetonitrile as an inhibitor. See Shapira and Arnon (1967) on antibody inhibitors. Papain may be inactivated by H₂O₂ generated by [[gamma]]-irradiation of H₂O- the active SH group being oxidized to sulfenic acid. (Lin et al 1975). See also Allison and Swain (1973).

Stability: Papain as a crystalline suspension is stable at 5°C for 6-12 months. Stabilizing agents are EDTA, cysteine and dimercaptopropanol.

To enhance stability as well as solubility it may be advantageous to convert crystalline papain to its mercury derivative (Brubacher and Bender 1966).