What is Papain?

Papain is an enzyme with proteolytic functions, classified as a cysteine protease. It belongs to a superfamily and consists of a single polypeptide chain containing three disulfide bonds and a thiol group essential for its enzymatic activity. Papain is derived from green papaya (see Figure 1), and its activity depends on the ripeness of the fruit. This enzyme plays a crucial role in various biological functions, such as hydrolyzing short peptides, proteins, esterified amino acids, and amide bonds, making it useful in both medical and food applications. It specifically cleaves positively charged amino acid peptide chains, mainly targeting lysine, arginine, and phenylalanine residues.

Molecular Weight: 23,406 Da
Optimal pH for Activity: 6.0-7.0
Optimal Temperature for Activity: 65 °C
Isoelectric Point (pI): 8.75 to 9.55
Spectral Properties: λmax: 278 nm
Extinction Coefficient: E1% = 25, EmM = 57.6 (280 nm)

Inhibitors: E-64, cysteine dihydrochloride, trypsin inhibitors, ibuprofen, and leupeptin trifluoroacetate.

Unit Definition: Under conditions of 25 °C and pH 6.2, one unit hydrolyzes 1.0 µmole of N-α-benzoyl-L-arginine ethyl ester per minute.

Source: Isolated from green papaya.

Specificity

Papain can digest a wide range of protein substrates, with a broader spectrum than trypsin. It exhibits extensive specificity, cleaving peptide bonds adjacent to basic amino acids, particularly leucine or glycine. Additionally, it can hydrolyze lipids and amide compounds. Papain preferentially targets amino acids with large hydrophobic side chains at the P2 position and does not accept valine at the P1' position.

Solubility and Solution Stability

Papain is soluble in water at a concentration of 10 mg/ml. Prior to use, it should be diluted in a buffer containing 5 mM L-cysteine. Its activators/stabilizers include EDTA, cysteine, and dithiothreitol. While papain solutions have good temperature stability, their stability is pH-dependent. Under acidic conditions, such as pH below 2.8, papain’s activity significantly decreases. In solution, papain loses approximately 1-2% of its activity daily, possibly due to self-degradation and/or oxidation. A common inactive form of papain obtained during isolation is a mixed disulfide formed between the active site thiol and free cysteine. Papain solutions remain stable in several denaturing agents, retaining full activity even after recrystallization with 70% methanol and 8M urea. However, treating papain with 10% trichloroacetic acid or 6M guanidine hydrochloride results in a significant loss of activity.