[5] Chymotrypsinogens—chymotrypsins

Diisopropylphosphorofluoridate reacts with trypsinogen and chymotrypsinogen and inhibits the potential activity of both zymogens. The reactions follow pseudo first-order kinetics and proceed approximately four orders of magnitude slower than diisopropylphosphorylation of the corresponding enzymes. Correlation of initial rates of inactivation with incorporation of the reagent indicates that zymogen inactivation results from incorporation of 1 mol of organic phosphate per mol of protein. Peptides isolated from the active-site region of trypsinogen account for more than 60% of the label originally present in the ['4Cjdiisopropylphosphoryl zymogen. It is concluded that loss of activation of trypsinogen is due to alkylphosphorylation of Ser183. It is proposed that reduced reactivity of the zymogen, as compared to the enzyme, primarily reflects inefficient binding of substrates and inhibitors, and that Ser183 of the active site exists in trypsinogen in an activated state.Diisopropylphosphorofluoridate (DFP) inhibits serine proteases by alkylphosphorylation of a specific serine residue of the active site (1). In contrast, and by definition, the parent zymogens are believed to be unreactive toward this and other active-site reagents (2-4). Recently, Robinson (5) reported that carbamylation of the a-amino group of E-guanidinated trypsin destroys esterase and amidase activity without abolishing reactivity toward DFP. Control experiments with native and guanidinated trypsinogen revealed unexpected reactivity of the zymogens toward DFP. It was concluded, therefore, that in trypsinogen (Robinson, N. C., Neurath, H. & Walsh, K. A., submitted for publication), as in trypsin (6), a serine residue of the actives site had been alkylphosphorylated.The present investigation was undertaken to measure the rate of zymogen inactivation, to confirm the site of interaction between DFP and trypsinogen, and to extend the applicability of these findings to chymotrypsinogen. 4-fold with 1.0 M sodium formate (pH 3.0) in order to quench the reaction with DFP. Excess DFP was removed from each aliquot by gel filtration on 1.5 X 6.0-cm columns of Sephadex G-25 equilibrated with 0.1 M sodium formate (pH 3.5). Protein concentration was determined by measuring the absorbance of each aliquot at 280 nm.Suitable portions of the desalted aliquots were removed, assayed for activatability, and counted on a scintillation counter. Trypsinogen (0.5 rng) in 1.0 ml of 0.1 M sodium formate (pH 3.5) was activated with 50 units of the acid protease from Aspergillus oryzae as described by Robinson (5).After 1 hr. the activation mixture was assayed for tryptic activity toward N-a-benzoyl-L-arginine ethyl ester as described by Walsh and Wilcox (7). "Activatability" was expressed in units of tryptic activity per mg of trypsinogen.Chymotrypsinogen (0.1 mg) was activated with 0.27 mg of trypsin after dilution to 2.0 ml with 1.0 M Tris HC1 (pH The oxidized protein (150 mg) was incubated with trypsin (3 mg) at 40°while the pH was maintained at 8.0. After base u...

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“…After 80 min chymotryptic activity was measured with N-a-acetyl-L-tyrosine ethyl ester as described by Wilcox (8). The (10).…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Bovine Trypsinogen and Chymotrypsinogen by Diisopropylphosphorofluoridate

Morgan

Robinson

Walsh

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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“…This is not straightforward unless the solutions are in pure water. Amino acid sequence data are available: ribonuclease A [lo]; lima bean trypsin inhibitor (composition only [l 13); hen lysozyme [12]; catalase [13]; a-lactalbumin [14]; chymotrypsinogen A and a-chymotrypsin [15] [39] were used with the carbohydrate data of [40] as representatives of IgG macromolecules.…”

mentioning

confidence: 99%

Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences

Perkins¹

1986

Eur J Biochem

561

268

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A wealth of accurate amino acid compositions for proteins and glycoproteins is presently available due to modern, rapid means of sequencing proteins and nucleic acids. In principal, macromolecular physical properties are more accurately and easily determined from these compositions in comparison to the use of classical biochemical techniques. The calculation of partial relative molecular masses M , is an obvious example of this. The calculation of macromolecular volumes V and partial specific volumes V is required in a range of applications: scattering density, matchpoint and molecular mass control measurements in X-ray and neutron solution scattering; molecular mass determinations by ultracentrifugation; constraints for use in low-resolution modelling of macromolecular shapes; packing analyses of amino acid and carbohydrate residues by solution or crystallographic studies. Starting from the classical 1943 Cohn and Edsall publication, several tabulations of residue volumes for amino acid and carbohydrates have been reported [l -71. These volumes can be summed on the basis of accurate amino acid and carbohydrate compositions to give V and V. In the present study, new compilations of amino acids and carbohydrate residue volumes are derived from small-molecule crystal studies. These are critically compared with previous compilations [I -71. The ability of these residue volumes to predict partial specific volumes and neutron scattering densities for proteins and glycoproteins is in turn critically compared with experimental data, bearing in mind that the partial volume is the particle volume corrected for hydration, solute binding and electrostriction effects. This clarifies the application of these calculations at a phenomenological level with the use of accurate composition data for not only solution scattering studies, but also for more general biophysical and biochemical Correspondence to S. J. Perkins, Kennedy Institute, Bute Gardens, Hammersmith, London W6 7DW, England applications. It is to be noted that these calculated V values are only valid for use in two-component solutions; in multicomponent solutions, such as in the presence of high concentrations of denaturants or electrolytes, considerable changes due to interactions with the solvent or with ligands will occur, and in such cases the calculations will generally fail [8]. Finally, the calculations of V and matchpoint data are correlated with protein hydration concepts on the basis of observed protein-bound water molecules from macromolecular crystal studies. This enables the macromolecular volume to be interpreted in molecular terms of (a) apparent changes induced by protein-bound water in densitometric studies and (b) the 'dry' molecular volume as visualised by neutron scattering studies.Macromolecular concentrations are conveniently determined in a wide range of applications by absorbance measurements at 280 nm on the basis of the absorption coefficients A:& Cm. The ability to calculate A:& ' cm from accurate amino acid sequences would be more straig...

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“…1). [15]. The quantity of the protein bound to liposomes was determined following its catalytic activity with ATEE as specific substrate…”

Section: Liposome Oxidation and A-chymotrypsinmentioning

confidence: 99%

Immobilization of α‐chymotrypsin on sucrose stearate‐palmitate containing liposomes

Богданов¹,

Klibanov²,

Торчилин³

1984

FEBS Letters

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[5] Chymotrypsinogens—chymotrypsins

Cited by 200 publications

References 39 publications

Inactivation of Bovine Trypsinogen and Chymotrypsinogen by Diisopropylphosphorofluoridate

Inactivation of Bovine Trypsinogen and Chymotrypsinogen by Diisopropylphosphorofluoridate

Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences

Immobilization of α‐chymotrypsin on sucrose stearate‐palmitate containing liposomes

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