Identity confirmation by PMF MALDI-TOF analysis of purified papain

LLERENA SUSTER, C.R.; TREJO, S.; OBREGÓN, W. D; MERCERAT, J. R; LOPEZ, L.M.I.; PRIOLO, N. S; MORCELLE, S.R

Rosario

Congreso; VII Simposio Nacional De Biotecnología Redbio; 2009

Papain (EC 3.4.22.2) is the most acidic peptidase from latex of Carica papaya fruit, and is considered the prototype of a very important group of cysteine peptidases. The enzyme was purified up to mass spectrometry (MALDI TOF) homogeinity by salt precipitation in presence of sodium tetrationate to preserve enzymatic activity. Refined crude extract preparation obtained in our laboratory, as well as a commercial crude preparation, were used as source for protease purification. The best condition found for this purification was by means of addition of 30 mM sodium tetrationate in the crude starting material. This was confirmed by conventional techniques such as SDS PAGE, isoelectric focusing and zymogram both in broad and alkaline pH range, and by cation exchange chromatography. Commercial pure papain was used as standard in all these assays for a rapid comparison. Confirmation of the identity of the purified plant peptidase was afforded by peptide mass fingerprint (PMF). This consisted in a fast and efficient method to identify papain from the other three peptidases contained in papaya latex (chymopapain, caricain and glycyl endopeptidase), which are very similar in other biochemical aspects, such as molecular weight and pI, the most common features to determine the progress of protein purification. On the other hand, the purification procedure demonstrated to be simple and provided pure papain in good yields to be applied in different processes of food, pharma and cosmetic industries, as well as some laboratory protocols and medical treatments.