Chymotrypsinogen chromatography

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. 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...

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Figure 6. Aqueous size exclusion chromatography of the APPL derived from S. badius. Ferritin, aldolase, ovalbumin, chymotrypsinogen A and acetone were used as standards.

Figure 2.9 Hydrophobic-interaction chromatography of proteins. (A) Ammonium sulfate gradient from 2.16 to 0 M (B) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 10 mM, respectively (C) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 20 mM, respectively (D) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 40 mM, respectively. Chromatography conditions column, silica-bound polyether, 10 cm x 4.6 mm I.D. temperature, 25°C flow rate, 1 ml/min gradient, linear for 30 min background buffer, 50 mM phosphate, pH 6.5. Peaks a, cytochrome c b, ribonuclease A c, /3-lactoglobulin A d, lysozyme e, ovalbumin f, a-chymotrypsinogen A g, fetuin. (Reprinted from Ref. 45 with permission.)...

Fig. 1. Chromatography of bovine pancreatic juice on DEAE-cellulose (anionic proteins) and Amberlite IRC-50 (cationic proteins) (1). RNAase, ribonuclease ChTg-a, chymotrypsinogen A Tg, trypsinogen ProCp-B and Cp-B, procarboxypeptidase B and carboxypeptidase B DNAase, deoxyribonuclease ProCp-A, procarboxypeptidase A.

Fig. 4. Chromatography of dog pancreatic juice on DEAE-cellulose (13, 14). The column is equilibrated with 0.005 M phosphate, pH 8.0 and eluted by a concentration gradient of phosphate indicated in the figure by a straight line. (1) Unfractionated cationic proteins (2) amylase (3) lipase (4) deoxyribonuclease (5) anionic chymotrypsinogen (6), (9), and (10) carboxypeptidase A and its precursor (7) and (8) carboxypeptidase B and its precursor. Ordinates on the left, optical density of the fractions at 280 m/ . Ordinates on the right, molarity of the phosphate. Abscissas, volume of eluate expressed in number of interstitial volumes of the column.

Keller and Cohen (36) also subjected to chromatography acidic extracts of cattle pancreatic microsomes and ribosomes. In microsomes they found the expected amounts of all enzymes which are known to be stable in acid, viz., chymotrypsinogen A, trypsinogen, deoxyribonuclease, and ribonuclease. The amounts of chymotrypsinogen B were abnormally low and ribonuclease B was perhaps not present. The results concerning ribosomes were made somewhat uncertain by the ability of these particles to incorporate proteins from the medium. Nevertheless, a series of characteristic enzymes could be isolated from what appears to be the very site of their biosynthesis. 

The second, anionic bovine chymotrypsinogen (chymotrypsinogen B), crystallized by Laskowski et al. (46), was initially thought to be of minor importance. This impression probably arose because of heavy losses during purification. Direct chromatography (1) proved later that the amounts of chymotrypsinogens A and B in bovine pancreatic juice were in fact very similar. Therefore it became important to compare as closely as possible the structures and modes of activation of two chymotrypsinogens synthesized by the same species. 

Fig. 11. Second chromatography of porcine chymotrypsinogen A and trypsinogen (85). Both elutions are performed with buffers of constant composition (equilibrium chromatography). On the left chymotrypsinogen A (specific activity, 2.9). CM-cellu-lose column equilibrated and eluted with 0.03 M citrate, pH 5.0. On the right trypsinogen (specific activity, 0.35-0.37). CM-cellulose column equilibrated and eluted with pH 6.0 buffer 0.015 M in citrate and 10 < M in DFP. Ordinates and abscissas are the same as in Figs. 9 and 10. Solid line, activity. Dotted line, proteins.

Bovine procarboxypeptidase B has been purified from pancreatin extracts (50). However, after a twenty-fold purification according to the technique described, the preparation still contains large amounts of chymotrypsinogen B (52). Final purification must therefore involve chromatography as well as fractional precipitations and extractions. 

Anion-exchange chromatography on manbranes, disks, and rods bearing mainly quaternary amino groups or DEAE groups as ligands has been used for the separation of serum proteins, microbial proteins and enzymes, membrane proteins, cytokines, or nucleic acids [40,69,249-254]. BSA and HSA, a-chymotrypsinogen, lysozyme, trypsin inhibitor, cytochrome c, ovalbumin, a-lactalbumin, conalbumin. 

---