Assessment of the Proteolytic Activity of α-Chymotrypsin Immobilized on Colloidal Particles by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Physisorption of enzymatically active chymotrypsin on titania colloidal particles

“…This contrasts literature data for TiO 2 with anatase crystal structure, which has been found to inactivate chymotrypsin by oxidative damages at least after exposure to ultraviolet light [48,49]. In addition, autolysis of chymotrypsin, which has been reported to take place during incubations at around pH 8 [50][51][52], appears not to be involved in the observed loss of chymotrypsin activity from the supernatant, consistent with recently published data under similar conditions [53].…”

“…Concerning potential technical applications of chymotrypsin that is immobilized by physisorption to titania particles it is important to investigate in future studies the stability and reusability of the particle-bound enzyme. For alumina and silica colloidal particles we have recently reported such data [53] and have demonstrated reusability of the immobilized enzymes by determining the proteolytic activity of the particle-bound enzyme in three consecutive 24 h lysozyme digestion steps. In addition, even after 7 weeks storage at room temperature, the immobilized chymotrypsin was still active to digest lysozyme as a substrate.…”

Atomistic Details of Chymotrypsin Conformational Changes upon Adsorption on Silica

“…We have found that chymotrypsin loses part of its helical content, with minor perturbation of its overall tertiary structure, associated with changes in the aromatic interactions, after adsorbing on silica. The observed structural changes could directly contribute to the experimentally measured decrease of chymotrypsin’s enzymatic activity in the adsorbed state. , The reported chymotrypsin case, however, should be considered as a proof of concept. We believe that the joint methodology introduced in this paper has a direct general applicability to the field of biomolecule–inorganic surface interactions.…”

“…The observed structural changes could directly contribute to the experimentally measured decrease of chymotrypsin's enzymatic activity in the adsorbed state. 16,17 The reported chymotrypsin case, however, should be considered as a proof of concept. We believe that the joint methodology introduced in this paper has a direct general applicability to the field of biomolecule -inorganic surface interactions.…”

“…How these changes can be predicted, described and correlated to physicochemical features of the material is however still unclear. Protein denaturation is commonly induced by adsorption on hydrophobic surfaces, whereas hydrophilic surfaces in general better preserve the native protein structures. − However, even small conformational changes can reduce the enzymatic activity, as frequently observed on functionalized oxide nanoparticles of alumina, titania or silica. , The present work aims at giving a precise, atomic-level insight into the conformational changes that a well-known enzyme (chymotrypsin) experiences when it adsorbs on the surface of a widely employed oxide, amorphous silica (SiO 2 ).…”