Papain, Lyophilized

Blue Ice

23

Supplied as a lyophilized powder prepared from a 2X crystalline suspension. It is recommended that the enzyme be 0.22 micron filtered after dissolution and prior to use.

Proteolytic enzymes are widely used in cell isolation. With some tissues papain has proved less damaging and more effective than other proteases. Papain is a sulfhydryl protease from Carica papaya latex. 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). Papain has wide specificity. It will degrade most protein substrates more extensively than the pancreatic proteases. It is also an esterase. The action of papain on leucine methyl ester produces an insoluble polyleucine peptide. Papain breaks down the intercellular matrix of cartilage.

Papain is a single peptide chain of 211 residues folded into two parts that form a cleft. A three-dimensional structure has been indicated. The molecule has one free SH group which is functional. There are seven subsites, each is capable of accommodating a single amino acid residue of a peptide substrate.

Activation:
To insure full activity, the enzyme should be incubated in a solution containing 1.1mM EDTA, 0.067mM mercaptoethanol, 5.5mM cysteine-HCl for 30 minutes.

Activity:
≥14 units per mg protein.

Unit Definition:
1 unit hydrolyzes 1umole of benzoyl-L-arginine ethyl ester per minute at 25ºC, pH 6.2, after activation in a solution containing 1.1mM EDTA, 0.067mM mercaptoethanol and 5.5mM cysteine-HCl for 30 minutes.

Quality Control:
SDS-PAGE

Optimum pH: 6.0 - 7.0
Extinction coefficient: = 25.0
Isoelectric point: pH 9.6
Activators: Papain is activated by cysteine, sulfide, sulfite, etc. It is enhanced when heavy metal binding agents such as EDTA are also present. N-bromosuccinimide enhances the activity.
Inhibitors: Substances which react with sulfhydryl groups including heavy metals, carbonyl reagents, aldehydes and benzoylamidoacetonitrile. See Shapira and Arnon (1967a and b) on antibody inhibitors. Papain may be inactivated by H2O2 generated by gamma-irradiation of H2O- the active SH group being oxidized to sulfenic acid.

Storage and Stability:
Lyophilized powder may be stored at 4°C. Stable for 6 months after receipt. Reconstitute with sterile buffer. Store at 4°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.