Trypsin titration

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

To the remainder of the casein solution add 0 5 to o 8 g. of finely powdered commercial trypsin, shake to dissolve, and place in a thermostat (or in an incubator) at 40 . After 15 minutes, remove 25 ml. and add a few drops of phenolphthalein it will now be found that the solution remains colourless. Run in carefully Mj 10 NaOH solution until the colour of the solution is just pink, add 5 ml, of neutralised formalin and then titrate against Mj 10 NaOH solution until the pink colour is just restored note the amount required. Remove fiirther quantities (rf 25 ml. at intervals which must be determined by the speed of the reaction. The following will probably make a suitable series i, 2, 3,... 

To the remainder of the gelatin solution, add 0 5 to o 8 g. of finely powdered commercial trypsin and incubate at 40 . Carry out the formaldehyde titration on 25 ml. samples at intervals as above. 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

Even though, the immobilization procedure should be engineered to maximum retained activity of immobilized enzyme, it is difficult to measure the amount of active enzyme on the carrier without an active site titration. However, this has been done in the case of immobilized trypsin, although only covalent immobilized. (Daly and Shih, 1982)... 

Associated with the problem of active-site titration is the question of the location of the active site in the three-dimensional structure of the protein. As a prelude to this investigation, a study is needed to indicate which amino acid residues in the overall peptide sequence are in the active site. The active site is defined as the location of the enzyme catalysis thus, the substrate complexes at the active site prior to the catalytic process. Addition of a substrate will, therefore, protect the enzyme against reagents, such as inhibitors, which react at the active site. Of course, the active site may include amino acid residues from distant parts of the peptide chain for example, both serine-195 and histidine-57 are in the active site of a-chymo trypsin. 

Talhout, R., Villa, A., Mark, A. E., and Engberts, J. B. F. N. (2003). Understanding binding affinity a combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin. Journal of the American Chemical Society 125, 10570-10579. 

Determination of the operational molarity of solutions of bovine O-chytnotrypsin, trypsin, thro mb to and factor Xa by spectre flu orometric titration. Biochem. J. 131 107 moth. 

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. 

A-Spectrum of Tyr and Phe shows complex pH-dependenoe trypsin digestion — A-spectrum Discrimination between individual abnormal Tyr s Perturbation of spectrum of protein-coupled anthracene by acid titration... 

A stock solution of trypsin (Sigma T-8642 or equivalent) is first standardized using the substrate p-nitrophenol-p -guanidobenzoate HCl (Sigma N-8010). By the initial burst principle (5) one can titrate the active site of trypsin and c culate the uM amount of active trypsin introduced into the rSLPI inhibition assay. By knowing the molecular weight of the trypsin (source dependent) the uM amount of rSLPI present can be deduced from the 1 1 stoichiometry of the reaction. 

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

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++. 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

X 10 moles trypsin per liter fluid volume. To demonstrate the feasibility of using the Ford method to determine the active-site of our immobilized enzyme systems, trypsin CVB-PHEMA-PABS-carbamate was treated in a circulation reactor with NPGB and the titration is Illustrated in Figure 4. The amount of p-nitro-phenol produced by the burst is equal to the amount of the active immobilized trypsin which, for this particular system, turns out to be 31% of the total bound enzyme. Active-site titrations of soluble trypsin were performed according to Chase and Shaw (16), and the active molecules for free trypsin was found to be 70% of the total protein involved. Consequently, the retention of active molecules for the immobilized enzyme was calculated 45%. The specific activity is 17% (Table III) for the same system so the efficiency of the system, based on the actually available active sites, was 38%. Thus, 62% of the initially active trypsin bound has lost its activity upon binding. 

Figure 4. Active site titration recorder trace of 17.0 mg trypsin CVB hydrogel sulfonyl carbamate (If) being titrated by 19.75 mL of 0.12 mM NPGB in pH 8.3 veronal buffer burst, 0.060 A.

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. 

T. Chase, Jr. and E. Shaw. Comparison of the esterase activities of trypsin, plasmin and thrombin on guanidino-benzoate esters. Titration of the enzymes. Biochemistry 8 2212 (1969). 

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