Chymotrypsin chromatography

The cysteinyl residue in the core was oxidized with performic acid according to the method of Hirs ( ) and the oxidized core was digested with chymotrypsin for 8 hours at room temperature in a pH stat at pH 8.9. Enzyme equal to 0.5% of the substrate by weight was added at 0 and at 2 hr. The chymotryptic peptides were also separated by Dowex 50-X4 chromatography. 

These high performance size exclusion separations of alpha-chymotrypsin SI and myosin light chains compare favorably with those achieved by ion exchange chromatography but require only a fraction of the time to accomplish. Furthermore, the very short retention times allow for separation of these labile proteins at room temperature, whereas operation at 0-40 C would otherwise be mandatory to avoid the loss of enzymatic activity. 

SEC is a useful tool for monitoring enzyme reactions, as seen in Figure 4.21, where the speed of decomposition of /Mactoglobulin by a-chymotrypsin is shown. SEC is widely used for purification of proteins, but the separation is due only to the difference in molecular mass. Therefore, ion-exchange liquid chromatography is combined with SEC to improve the selectivity. 

Figure 4.21 Monitoring of an enzyme reaction using size-exclusion liquid chromatography. Column, TSK GEL G3000SW, 60 cm x 7.5 mm i.d. eluent, 0.07 M potassium phosphate buffer containing 0.1 M potassium chloride flow rate, 1 ml min-1 detection, UV 280 nm. Peaks 1, fl-lactoglobulin 2, a-chymotrypsin, and 3, decomposed products.

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

Shivraj, B., Rao, H. N., and Pattiraman, T. N. (1982). Natural plant inhibitors. Isolation of a trypsin/a-amylase inhibitor and a chymotrypsin inhibitor from ragi (Eleusine coracana) grains by affinity chromatography and study of their properties. J. Sci. Food Agric. 33,1080-1091. 

Receptor type DNA-cellulose chromatography (mM KC1 required for elution) native after chymotrypsin Molecular weight of hormone-labelled polypeptide native after chymotrypsin ... 

Fig. 8. Purification of chain C by chromatography on Sephadex G-50 (79). Sepha-dex G-50 column (1.8 X 35 cm) equilibrated with 0.05 M HCl. Lyophilized aqueous extract of 30 mg of DFP-inhibited, performic acid-oxidized a-chymotrypsin (A4) is dissolved in 1 ml 0.05 M HCl, put into the column, and eluted (5 ml/hr) by 0.05 M HCl. Solid line, absorption of the fractions at 280 mu. Dotted line, absorption at 230 mil. Ordinates, optical density. Abscissas, volume of eluatein milliliters. A, chain A C, chain C.

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