PubMed · 10503446
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M Katoh. 1999. [Chymotrypsin].. https://pubmed.ncbi.nlm.nih.gov/10503446/
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For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degrees C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.
We studied the inactivation of trypsin and alpha- and beta-chymotrypsin by passage of droplets of tridecane though their aqueous solutions. The mechanism involves contact with the interface, because the loss of activity is proportional to the total area exposed. The rates of inactivation vary up to fivefold over the pH range 3 to 10. However, there is no clear maximum at the isoelectric point (pI) of each enzyme, where the amount of protein adsorbed is usually found to be highest. This is probably because, at the pI, there is also a minimum in structural alteration on adsorption. There may be a weak correlation with pH effects on foamability of the enzyme solutions, a parameter reported to reflect the "hardness" of different proteins, which controls their interfacial unfolding. The pH dependence of both inactivation and hardness cautions against attempts to correlate inactivation of different enzymes with a single value of a parameter such as adiabatic compressibility. There is no correlation between the effects of pH on interfacial inactivation and those reported in the literature on irreversible inactivation in concentrated urea or at high temperature.
A study of various 2-[(alkylsulfonyl)oxy]-6-substituted-1H-isoindole-1,3-diones' inhibition of chymotrypsin compared to inhibition of HLE reveals that acylamino substitution in the 6-position increases selectivity and potency of these inhibitors for HLE. The best HLE inhibitor in this series was 6-(methylglutaryl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-1,3-di one with a kobs/[I] = 220,000 M(-1) s(-1).