Papain purification method

Claradiastase and trypsin in phosphate buffer have been used for hydrolysis of sample matrix for the determination of supplemental folic acid (27). Jacoby and Henry (26) further modified the method of Hoppner and Lampi (41) for folic acid by HPLC, in which the folic acid added to infant formulas and liquid medical nutritionals is quantitatively extracted with the aid of bacterial protease and papain. One disadvantage of the enzymatic extraction method was the large number of UV-absorbing compounds that are formed during enzymatic hydrolysis. These can interfere in the quantitation of the folate peaks. Thus, addition of a-amylase and protease to enhance extraction has not been common practice for methods involving HPLC. However, Pfeiffer et al. (13) suceessfiilly used triple-enzyme treatment prior to HPLC analysis with a purification method based on affinity chromatography. 

The interest in papain increased enormously after the publication by Kimmel and Smith D ] of a modification of the purification procedure of Balls and Lineweaver [5j. This modification permitted the isolation of pure papain from papaya dried latex and was used for many years as the standard method for the production of papain. The crystalline papain of Kimmel and Smith consists of three components active papain, reversibly oxidized papain, and irreversibly oxidized papain. Reversibly oxidized papain can be converted into active papain by reduction of the active-site thiol by low molecular weight thiols [10], sodium borohydride [11], or CN [12]. In active papain, the Cys-25, which is essential far catalytic activity, is present in a reduced form, while in reversibly oxidized papain the Cvs-25 forms a mixed disulfide with cysteine. Drenth et al. reported that in irreversibly oxidized papain the Cys-25 has been oxidized to the sulfuric acid... 

The patent [24] described the method of production of hyaluronan fractions with the average molecular mass from 250000 to 350000Da. The proteins, which were still found in the extracts, were hydrolysed with papain, and then the resulting solution underwent ultrafiltration through a membrane. In addition to purification, ultrafiltration afforded separation from the fraction of 30 000Da and lower, since such a low HA molecular mass could activate inflammation processes for when apphed parenterally. The membrane can hold the HA fractions above 30000. 

To escape the divalent character of immunoglobulin, Fab fragments which possess only one binding site can be used. Two different methods were described for purification of monovalent F.b fragments from IgG. One is a digestion by papain (Porter, 1959) at an enzyme-to-IgG final ratio of 1 25... 

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