Methanol Papain

Due to the relative stability of the niacin vitamers, either acid or alkaline hydrolysis can be used to convert nicotinamide to nicotinic acid for quantitation of both vitamers as nicotinic acid (9,44). Acid hydrolysis is used to quantitate biologically available niacin. Alkaline hydrolysis releases both the biologically available and the unavailable vitamers and provides an estimate of the total niacin content. Because alkaline hydrolysis is much faster than acid hydrolysis, the latter is usually supplemented with enzymatic hydrolysis. The most common enzymes are takadiastase, papain, and clarase. On occasion, organic solvents such as methanol have been used to extract free nicotinic acid. 

Figure 9.28 HPLC profile of papain-catalyzed B AEE hydrolysis product. Conditions column, juBondapak CN eluent, 0.05 M ammonium acetate-methanol (85 15) flow rate, 2.0 mL/min detector, 254 nm at ambient temperature (From Chen et al., 1982.)...

Studies of blistering (acantholysis and vesiculation) show that disc proteins are attacked by papain, trypsin, and by reagents that split sulfur or hydrogen bonds (36, 37). Desmosomes and cells are also separated by extraction of calcium ions (35, 37), presumably from proteoglycans. Desmosomes are completely dissolved in vitro by chloroform methanol mixtures (38) that dissolve proteolipids (39). The separated cells become reaggregated after evaporation of the solvent leaving an intercellular deposit with normal appearance by electron microscopy (38). 

Arad et al. (1990) simulated the reaction sequence of papain by constructing several enzyme-substrate models with molecular mechanics and following reaction paths with semiempirical quantum mechanics. AMBER force field (Weiner et al., 1986a) was employed for the construction. AMI (Dewar et al, 1985) results for proton affinities of the modeled molecules were compared to 4-31G and to experiments. AMI underestimates the proton affinities of methanethiol and of imidazole but overestimates the proton affinity of methanol. However, the proton transfer reactions from methanol to imidazole and from methanethiol to imidazole are overestimated by only 6 and 11 kcal/mol, respectively, and PT from imidazolium to formamide is underestimated by 6 kcal/mol. 

A few examples of enzyme-catalysed peptide synthesis will suffice to illustrate its power and flexibility. As indicated above, proteinases are usually stereospecific for amino-acid residues at positions Pj and Pj. Stereospecificity can sometimes be relaxed in solutions containing organic solvents. Thus papain affords quite high yields of protected dipeptides when it is incubated with Z—Gly—OEt and H—d— Ala—OEt, H—d—Leu—OMe, H—d—Phe—OMe or H—d—Val—OMe in aqueous methanol. 

White powder. Soluble in 80% ethanol. 90% methanol and 75% acetone. Practically insol in ether. Stable at pH 1.0-12.0. Adsorbed on Amberlite IRC 50 (H-form). Acts like a high-molecular weight polypeptide. Attacked by papain. 

The cell membranes are usually isolated from keratins after digestion of the hair with an enzyme and a reducing agent such as papain in the presence of dithiothreitol and then extracted with a lipid solvent system such as chloroform/methanol see the procedure by Swift and Bews [81,82]. 

An equimolar soln. of glycine isopropyl ester hydrochloride and D-alanine methyl ester in methanoll 3 M tris(hydroxymethyl)aminomethane containing a little 2-mer-captoethanol adjusted to pH 9.5 with 10 MNaOH, 0.5 eqs. of N-benzyloxycarbonyl-phenylalanine methyl ester and a little (crude) papain added successively, the mixture shaken for 1 min, methyl isobutyl ketone added, and stirred vigorously at room temp, for 12 h - Z-Phe-Gly-D-Ala-OMe. Y 57%. A 2-fold increase in yield was observed with methanol as cosolvent compared with dioxane. The method appears to be quite... 

Fragment condensations with papain have also been reported, for example enkephalin fragment coupling (2 + 3) was achieved in 50% 5tield using equimolar amounts of acyl donor (PhAc-Tyr-Gly-OMe) and nucleophile (H-Gly-Phe-Leu-0 Bu) af pH 9.0 in buffer with 20% methanol [26]. Similar yields were obtained in low water systems such as buffer containing acetonitrile 4% (v/v). 

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