Trypsin titration curves

For Questions 4.14-4.17, use the following information. You have isolated an unknown peptide whose titration curve is presented in Figure 4.16. The peptide absorbed ultraviolet light at 280 nm. Treatment with trypsin released free alanine and arginine. Treatment with chymotrypsin resulted in quantitative production of a neutral tripeptide and an alkaline dipeptide. 

Redox potentials have been determined for each of the steps of reduction of the trypsin-solubilized reductase (403) step 1, one electron consumed, Eo = —109 mV step 2, two electrons consumed. Eg = —276 mV and step 3, one electron consumed. Eg = —371 mV at pH 7.0, 26°. As expected, the redox potential of step 3 is more negative than the potential of the NADPH-NADP+ couple and was determined from the dithio-nite titration. The overall potentiometric—spectrophotometric titration curves could be very closely fitted with a computer-generated curve based on the assumptions of four one-electron reduction steps and octinction coefficients of 4.9 and 4.5 mM cm for the semiquinones, FliH and rijH the Eg values assumed for steps 2 and 3 were —270 and —290 mV. The precise fit was very sensitive to all of the assumptions (40 ) ... 

Titration curves of trypsin were obtained under a variety of conditions by Duke et al. (1952). The most noteworthy feature is a specific effect of calcium, which displaces the acid part of the titration curve to lower pH, and decreases the total number of groups which are titrated between pH 6 to 9. It is likely that the groups titrated between pH 6 and 9 in the absence of Ca " are a-amino groups, produced by self-digestion of the enzyme. The effect of Ca" " thus appears to result from a complex with the carboxyl groups of the protein, which stabilizes the anionic form of these groups so as to produce the displacement of the acid part of the titration curve. This complex is more resistant to self-digestion than the enzyme alone. 

Calcium ion has a very specific effect on trypsin (Gorini, 1951), and Mn++ and Cd++ act similarly. However, other bivalent ions such as Mg++, Ba++, Si, Co++, Cu", or Ni++ are without effect (Nord and Bier, 1953). Duke, Bier, and Nord (1952) found that calcium increased the acidity of the carboxyl groups of trypsin between pH 3.5 and 5, and concluded that this was due to chelation of Ca++ with carboxyl groups. Their titration curves for trypsin in the presence of CaCh, MgCh, and KCl are shown in Fig. 5. At pH below 3 the enhanced acidity due to Ca++ disappears, since chelation with undissociated carboxyl groups is impossible. Nanninga (1954) has made a similar observation in the titration of L-meromyosin in the presence of Mg++. 

Phase boundaries were also developed for p-lactoglobulin, chicken egg albumin, lysozyme, ribonuclease, and trypsin, all at r=100, a weight ratio at which polymer saturation appears to take place (see Discussion section). For each protein, pHcritical was converted to net negative surface charge (Zpr) per unit protein surface area (A), using potentiometric titration curves (26-31) and hydrodynamic radii (32) found in literature. Plots of surface charge density (Zpr/A) vs. I are shown in Figure 3. 

Streptomyces griseus trypsin [8] and a-lytic protease [9] are serine proteases with a solitary histidine residue. This histidine residue is believed to be involved in the catalytic mechanism via a charge-relay mechanism analogous to that in a-chymotrypsin. Figures 2 and 3 show the reactivity data for the histidine residue in a-lytic protease and S.G. Trypsin. The data for a-lytic protease approximately follow a titration curve... 

Fig. 5. Titration of trypsin in the presence of CaCh, MgCls, and KCl. From Duke, Bier, and Nord (1952), with difference curve added (bottom).

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