Ultraviolet spectral changes related to the enzymic activity of chymotrypsin

Chervenka, Charles H.

doi:10.1016/0006-3002(59)90442-1

Cited by 82 publications

(15 citation statements)

References 20 publications

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“…This has been attributed to a change in the environment of the tryptophyl groups. 15 The data reported here show that the changes in optical rotation and in optical density, which occur when solutions of DIP-CT and monoacetyl-CT are heated at pH 2.0, parallel each other. However, the polarimetric and spectrophotometric methods do not give the same results for the CT transitions.…”

Section: Methodssupporting

confidence: 53%

Evidence for Conformational Changes in α-Chymotrypsin-catalyzed Reactions. VI. Changes in Optical Rotatory Dispersion Parameters

Havsteen¹,

Hess²

1963

J. Am. Chem. Soc.

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Section: Methodssupporting

confidence: 53%

Evidence for Conformational Changes in α-Chymotrypsin-catalyzed Reactions. VI. Changes in Optical Rotatory Dispersion Parameters

Havsteen¹,

Hess²

1963

J. Am. Chem. Soc.

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“…Activation of the zymogen was obtained by overnight incubation at 4°C with bovine enterokinase (Boehringer) using an enzyme:substrate ratio 1:30 (w/w). To reduce autodigestion, this activation was performed at 4°C in the presence of Ca 2+ ions and the reversible chymotrypsin inhibitor ε‐amino‐n‐caproic acid (Chervenka 1959; Delaage et al 1968; Steffen and Steffen 1976; Sano et al 1988). The active chymotrypsin forms were further purified at 4°C using a XK‐16 column (Pharmacia) packed with 10 mL SBTI Sepharose resin (Sigma‐Aldrich) (Amneus et al 1976; Kasche et al 1977) in the polishing step.…”

Section: Methodsmentioning

confidence: 99%

A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

et al. 2004

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The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of ␣-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.

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“…This decrease has been reported to accompany the unfolding of CT and has been attributed to a change in environment of tryptophyl residues. 29 The thermodynamic parameters obtained by the two methods, [a ]365 and AD292, are in excellent agreement for monoacetyl-CT and DIP-CT, but differ in the case of CT. The details of the experiments on the temperature dependence of D292 are reported elsewhere.30 The thermodynamic parameters obtained from the temperature dependence of [a ]36r are listed in Table 1.…”

mentioning

confidence: 87%