Papain titration

Fig. 11.10 (A) Burst kinetics for release of the leaving group from a substrate of a-chymotrypsin. (B) Titration of papain with the irreversible inhibitor 4-toluenesulphonamidomethyl chloromethyl ketone using methyl benzoylglycinate as substrate.

Glazer and Smith (1961) have carried out a spectrophotometric titration of the phenolic groups of papain. Of the seventeen phenolic groups known to be present, eleven to twelve ionize normally (pKint = 9.8). The remainder ionize only upon denaturation, which takes place only slowly in the range of pH 12 to 13. 

Papain Three classes of Tyr ionization titration irreversible above pH 12 3 of 4 Tyr s ionize at pH <12.5 but not all reversibly Glazer and Smith (1961b)... 

FIG. 1 Turbidimetric titration curves (so-called colloid titration) of different papain solutions with a solution of potassium poly(vinylsulfate) at 2.5 mmol/L. The volume of titrant at the abrupt increase of turbidity (V, ) is given in the figure. At the point of incipient precipitation, the polymer/protein ratio is constant. (Reprinted with permission from Ref. 27. Copyright 1996 American Chemical Society.)... 

FIG. 6 Ratio of the sulfate groups to the protein mass at the onset of precipitation (colloid titration) upon titration of papain with a polyanion at low ionic strength. Circles poly(vinylsulfonate) triangles poly(styrene sulfonate) of low Mw (68.000) squares high Mw poly(styrene sulfonate). The fully ionized papain at low pH corresponds to 1.07 mmol/g of cationic residues. (Reprinted from Ref. 10 by courtesy of Marcel Dekker, Inc.)... 

Liberators of antithrombin and/or heparin cofactor. The latter proteins appear to be secreted by the liver, and result in hypocoagulability in thrombin titration tests and increase the sensitivity of the coagulation time and other tests to heparin. Papain releases antithrombin. ... 

Migliorini, M., Creighton, D.J. Active-site ionizations of papain. An evaluation of the potentiometric difference titration method. Eur. J. Biochem. 1986,156,189-92. 

In turbidimetric titration, a polyelectrolyte is added incrementally to another highly dilute polyelectrolyte solution and the intensity of the light scattered by or the turbidity due to complex or precipitate formation is measured. In this colloid titration, turbidity is recorded as a function of titrant volume and the end point indicated by maximum turbidity is used to ascertain the stoichiometry of complex formation as shown in Figure 56.5. Jiang and Zhu" used colloid titration to investigate PAA-gelatin complexation and Tsuboi et al." used it to study complexation of papain with potassium poly(vinylsulfonate) (KPVS). 

Since the NMR resonances of the carbonyl of thioesters are shifted (A 20 to 30 ppm), NMR spectroscopy should allow the direct monitoring of the formation and decay of a thioacyl intermediate. Using = O] N-benzoylimidazole (5 = 168.7 ppm), we were able to observe directly a thioacyl intermediate at 8 = 195.9 ppm in the presence of papain under the cryoenzymological conditions of -t C in 25 percent aqueous dimethyl sulfoxide. Moreover, the thioacyl species is clearly a productive intermediate since the decrease in its signal intensity was accompanied by an increase in the product resonances and by release of free enzyme (half-life, 96 minutes) determined by titration of its thiol group. The line width of the resonance at 195.9 ppm was 25 Hz [5,6]. 

Use the method most applicable to the antibody imder study. For instance, anti-D can he detected by agglutination of native (for IgM) or papainized (for IgG) D positive red cells. The culture supernatant may be tested undiluted or titrated the titre will give an indication of antibody concentration. Quantitation of total immunoglobulin concentration is not informative until the cells are cloned. 

Protease Papain Fabric Titration, water absorption assays Hydrolysis, increased hydrophilicity [53]... 

The proteolytic enzyme of papain is active in both acid and alkaline media. Bullock and Sen recommended casein as substrate for formol titrations. The importance of activation, preferably with cysteine hydrochloride, was established and the follov/ing assay proposed. 

In each of two flasks place 15 ml of casein solution and 30 ml of water, adjust the temperature to 60° and place in a water-bath at 60°. To one flask add 5 ml of the solution of papain and cysteine hydrochloride and to the other add 5 ml of a portion of the same activated enzyme solution previously boiled for three minutes and cooled. Maintain both at 60° for thirty minutes. Cool rapidly to room temperature and to each flask add 0-75 ml of 0 1 per cent phenolphthalein solution and 10 ml of formaldehyde solution previously neutralised to phenolphthalein. Titrate both liquids with G IN sodium hydroxide to a definite pink colour (pH 8-7). The difference between the two titrations should not be less than 4-5 ml or more than 6-0 ml. 

The descending portions of the curves for ki in Figure 26 must represent the titration of groups essential for papain activity. On the acid side of the curve, ki reaches a maximum near pH 5. The... 

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