Chymotrypsinogen

The polypeptide chain of chymotrypsinogen, the inactive precursor of chymotrypsin, comprises 245 amino acids. During activation of chymotrypsinogen residues 14-15 and 147-148 are excised. The remaining three polypeptide chains are held together by disulfide bridges to form the active chymotrypsin molecule. 

Figure 11.7 Schematic diagram of the structure of chymotrypsin, which is folded into two antiparallel p domains. The six p strands of each domain are red, the side chains of the catalytic triad are dark blue, and the disulfide bridges that join the three polypeptide chains are marked in violet. Chain A (green, residues 1-13) is linked to chain B (blue, residues 16-146) by a disulfide bridge between Cys 1 and Cys 122. Chain B is in turn linked to chain C (yellow, residues 149-245) by a disulfide bridge between Cys 136 and Cys 201. Dotted lines indicate residues 14-15 and 147-148 in the inactive precursor, chmotrypsinogen. These residues are excised during the conversion of chymotrypsinogen to the active enzyme chymotrypsin.

Wang, D., Bode, W., Huber, R. Bovine chymotrypsinogen A. X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution. /. Mol. Biol. 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

In a study of the temperature-induced reversible denaturation of the protein chymotrypsinogen,... 

The equilibrium constants determined by Brandts at several temperatures for the denaturation of chymotrypsinogen (see previous Example) can be used to calculate the free energy changes for the denaturation process. For example, the equilibrium constant at 54.5°C is 0.27, so... 

Eigure 3.5 presents the dependence of A.S ° on temperature for chymotryp-sinogen denaturation at pH 3. A positive A.S ° indicates that the protein solution has become more disordered as the protein unfolds. Comparison of the value of 1.62 kj/mol K with the values of A.S ° in Table 3.1 shows that the present value (for chymotrypsinogen at 54.5°C) is quite large. The physical significance of the thermodynamic parameters for the unfolding of chymotrypsinogen becomes clear in the next section. 

FIGURE 3.4 The dependence of AG on temperatnre for the denaturation of chymotrypsinogen. (Adapted from Brandts, J. K, 1964. The thermodynamics of protein denaturation. I. The denaturation of e.hymotryjosinogeti. femrntA of the American Chemical Society m-.429I-430I.)... 

FIGURE 16.16 Comparison of the amino acid sequences of chymotrypsinogen, trypsino-gen, and elastase. Each circle represents one amino acid. Nmnbering is based on the sequence of chymotrypsinogen. Filled circles indicate residues that are identical in all three proteins. Disnlfide bonds are indicated in yellow. The positions of the three catalytically important active-site residues (His, Asp °-, and Ser ) are indicated. 

Active a-chymotrypsin is produced from chymotrypsinogen, an inactive precursor, as shown in the color figure on page 530. 

Fig. 13. Relative sorption capacity of proteins by carboxylic CP Biocarb-T vs pH of solution 1) terrilytin, 2) insulin, 3) chymotrypsinogen, 4) pancreatic ribonuclease, 3) pepsin, 6) thymarine, 7) thermolysine, 8) haemoglobin, P) lysozyme. mma, — quantity of protein bonden on Biocarb-T by pHma (...

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Fig. 4. HPHIC of standard proteins on the weak hydrophobic columns. The SynChro-pack PROPYL column was 25x0.41 cm Poly (alkyl aspartamid)-silicas were packed into 20 x 0.46 cm columns. Sample 25 pi containing 25 pg of each protein in buffer A. Buffer A 1.8 mol/1 ammonium sulphate + 0.1 mol/1 potassium phosphate, pH 7.0. Buffer B 0.1 mol/1 potassium phosphate, pH 7.0. Gradient 40-min linear 0-100% buffer B. Flow rate 1 ml/min. Detection A220 = 1-28 a.u.f.s. Peaks a = cytochrome C, b = ribonu-clease A, c = myoglobin, d = conalbumin, e = neochymotrypsin, / = a-chymotrypsin, g - a-chymotrypsinogen A [48]...

In Fig. 11, separations of chymotrypsinogen A, cytochrome c and lysozyme on strong cation exchangers carrying SO3 groups by gradient elution are shown. 

Fig. 11a. Fractionation of (/) chymotrypsinogen A, (2) cytochrome C and (3) lysozyme on strong cation exchangers, a) Support Fractogel TSK 650(s)SP (conventional type) sample, 1 mg each flow rate, 1 ml/min column size, 150 x 10 mm T.D. Solvent A = 0.02 mol/1 phosphate, pH 6.0 solvent B = A + 1 mol/1 NaCl gradient, 0-10 min, 0% B 10-70 min, 0-100% B. b) Support Fractogel EMD 650(s)SO( — (tentacle type) conditions as in (a) [78]...

P9.2 The free energy change for the reaction between native and denatured chymotrypsinogen has been measured as a function of temperature and pressure. The reaction can be described as ... 

S. A. Hawley, Reversible Pressure-Temperature Denaturation of Chymotrypsinogen",... 

Fractionated ammonium sulfate precipitaion of a-chymotrypsinogen (further fractions contain deoxyribonuclease, chymotrypsinogen B, ribonuclease, trypsinogen). 

The proteases are secreted as inactive zymogens the active site of the enzyme is masked by a small region of its peptide chain, which is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin (autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells trypsin can then activate chymotrypsinogen to chymotrypsin, proelas-tase to elastase, procarboxypeptidase to carboxypepti-dase, and proaminopeptidase to aminopeptidase. 

Fig. 5. The approximate molecular mass determination of polygalacturonase [(0—0) - substrate 0.5% pectate, pH 4.6] and exopolygalacturonase [( — ) - substrate 1.0 pmol/ml of di(D-galactosiduronic) acid, pH 4.0] on Superose 12 column (FPLC device). Flow rate 0.5 ml/min. System 0.05 M phosphate buffer pH 7.0, 0.15 M NaCl. Standarts Ferritin (450 kDa), Katalase (240 kDa), Aldolase (158 kDa), Albumin (68 kDa), Albumin (45 kDa), Chymotrypsinogen A (25 kDa), Cytochrome C (12.5 kDa).

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

Chymotrypsin has also been utilized to promote debridement, as well as the reduction of soft tissue inflammation. It is also used in some opthalmic procedures, particularly in facilitating cataract extraction. It is prepared by activation of its zymogen, chymotrypsinogen, which is extracted from bovine pancreatic tissue. 

Mixture of proteins (Myoglobin, Ovalbumin, Lysozyme and Chymotrypsinogen) Purification Hydrophobic Interaction [52]... 

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

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