Figure 1.Isolated from Papaya (Carica papaya) Latex
Papain is a cysteine protease of the peptidase C1 family. Papain consists of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for activity of the enzyme.
Molecular weight: 23,406 Da (amino acid sequence)16
Optimal pH for activity: 6.0-7.0
Temperature Optimum for Activity: 65 °C22
pI: 8.75 17; 9.55 18 Spectral properties:
λmax: 278 nm 19
Extinction coefficient, E1% = 25 19
Extinction coefficient, EmM = 57.6 (at 280 nm) 20
Unit Definition: One unit will hydrolyze 1.0 µmole of N-α-benzoyl-L-arginine ethyl ester (BAEE) per minute at pH 6.2 at 25 °C.
Papain will digest most protein substrates more extensively than the pancreatic proteases. Papain exhibits broad specificity, cleaving peptide bonds of basic amino acids, leucine, or glycine. It also hydrolyzes esters and amides. Papain exhibits a preference for an amino acid bearing a large hydrophobic side chain at the P2 position. It does not accept Val at the P1' position.1
Figure 3.Pepsin and Papain Cleavage
Papain is soluble in water at 10 mg/mL. Immediately prior to use, the enzyme is typically diluted in buffer containing ~5 mM L-cysteine. Activation/stabilizing agents include EDTA, cysteine, and dimercaptopropanol.21
Although papain solutions have good temperature stability, the solution stability is pH dependent. Papain solutions are unstable under acidic conditions, i.e., at pH values below 2.8, there is a significant loss in activity. For the active enzyme in solution, the loss in activity is about 1-2% per day, probably as a result of autolysis and/or oxidation.
A common inactive form of papain obtained during isolation is a mixed disulfide formed between the active site sulfhydryl group of the protein and free cysteine.23
Papain solutions are stable to several denaturing agents, i.e., full activity is maintained after recrystallization in 70% methanol and in 8 M urea solutions. However, there is a significant loss in activity when papain is exposed to 10% trichloroacetic acid or to 6 M guanidine hydrochloride.