betadex and alpha-chymotrypsin

A diradical-platinum(II) complex was able to recognize the subtle difference in cavity size between β- and γ-cyclodextrin with on-off switching of intense near-infrared absorption. This provides a new probe for identifying the size of hydrophobic cavities, which has been successfully applied here to differentiate human serum albumin from α-chymotrypsin.

Topics: Aminobenzoates; beta-Cyclodextrins; Chymotrypsin; Coordination Complexes; gamma-Cyclodextrins; Humans; Hydrophobic and Hydrophilic Interactions; Nuclear Magnetic Resonance, Biomolecular; Platinum; Serum Albumin; Spectrophotometry

J-Aggregates of diprotonated 5,10,15,20-tetrakis(4-sulfonatopheny)porphyrin (H₄TPPS²⁻) were stabilized even in a neutral aqueous solution (pH 7.0) containing per-O-methylated β-cyclodextrin by binding to the surface of α-chymotrypsin (ChT). The large J-aggregates covered the active site of ChT and completely inhibited the hydrolysis of the peptides. However, enzyme activity was gradually restored with the dissociation of the J-aggregates attached to the protein surface to monomers. After the completion of dissociation of the aggregates, the enzyme activity was almost completely restored, though the structure of ChT significantly changed. Circular dichroism spectroscopy suggested that the microscopic structure at the active site of ChT was scarcely affected by the J-aggregates, but the binding of J-aggregates to ChT increased the content of the random coils in the enzyme. The present study showed a new type of effector for controlling the function of ChT.

Topics: Animals; beta-Cyclodextrins; Binding Sites; Catalytic Domain; Cattle; Chymotrypsin; Circular Dichroism; Enzyme Activation; Enzyme Inhibitors; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Models, Molecular; Pancreas; Porphyrins; Protein Binding; Protein Structure, Secondary; Solutions; Substrate Specificity; Time Factors; Water

The potential of cyclodextrins to stabilize alpha-chymotrypsin upon encapsulation in Poly(lactic-co-glycolic) acid (PLGA) microspheres using a solid-in-oil-in-water (s/o/w) technique was investigated. Two cyclodextrins, hydroxyl-propyl-beta-cyclodextrin (HPbetaCD) and methyl-beta-cyclodextrin (MbetaCD), one insoluble and the other soluble in methylene chloride, were used. The results demonstrate that HPbetaCD failed to stabilize alpha-chymotrypsin upon encapsulation. Specifically, 19% of the protein was aggregated and the specific activity of the enzyme was reduced to ca. 50% of that prior to encapsulation. In contrast, MbetaCD significantly decreased the formation of aggregates to 3% and the retained specific activity of the enzyme was approximately 90%. The co-lyophilization of alpha-chymotrypsin with MbetaCD prior to encapsulation was a requisite to preserve the protein stability in microspheres. Furthermore, MbetaCD prevented the loss of protein during the preparation of microspheres and the encapsulation efficiency was improved to 90%. Release experiments showed the use of MbetaCD modified the release profile: the burst release decreased from 54% (in the absence of the excipient) to 36%. The results suggest that MbetaCD might be a suitable excipient to improve protein stability in s/o/w encapsulation procedures.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Chymotrypsin; Drug Compounding; Emulsions; Enzyme Stability; Excipients; Lactic Acid; Methylene Chloride; Microspheres; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Solvents; Surface Properties; Water

Bovine pancreatic alpha -chymotrypsin was chemically modified with two different beta -cyclodextrin derivatives, named mono-6-formyl-beta-cyclodextrin and mono-6-succinyl-6-deoxy-beta-cyclodextrin. The modified enzymes contained approx. 3-5 mol of oligosaccharide/mol of protein, and retained full proteolytic and esterolytic activity. The optimum temperature for alpha -chymotrypsin was increased by 8 degrees C and its thermostability was enhanced by about 4-6 degrees C after modification. The conjugated enzymes were also more resistant to thermal inactivation at temperatures ranging from 45 to 55 degrees C. Additionally, the modified enzymes were 7-fold more stable against incubation at pH 9.0. The possible influence of supramolecular interactions on the thermal stabilization of modified alpha -chymotrypsins was also studied.

Topics: Animals; beta-Cyclodextrins; Cattle; Chymotrypsin; Cyclodextrins; Enzyme Activation; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Pancreas; Protein Conformation; Sensitivity and Specificity; Temperature

The purpose of this study is to investigate the mechanisms and thermodynamics of the interaction between hydroxypropyl beta-cyclodextrin (HPdetaCD) and [D-Trp6, des-Gly10] LHRH ethylamide (deslorelin), a peptide drug.. We used UV and fluorescence spectroscopy to study the interaction of HPbetaCD and deslorelin. Circular dichroism was used to study the conformational changes induced in deslorelin upon interaction with HP beta CD. The thermodynamics of the interaction of deslorelin and HPbetaCD was studied using isothermal titration calorimetry (ITC). We also determined the effect of HPbetaCD on the degradation of deslorelin by alpha-chymotrypsin.. UV and fluorescence spectroscopy indicated that HPbetaD induced a change in polarity of the environment surrounding the chromophores of deslorelin. Wavelength selective fluorescence indicated an increase in the fluorescence polarization of deslorelin with an increase in excitation wavelength in the presence of HPbetaCD suggesting that tryptophan is present in a media of reduced mobility. Circular dichroism studies indicated that HPbetaCD stabilizes the conformation of deslorelin. In addition, ITC indicated an exothermic reaction between deslorelin and HPbetaCD with a low enthalpy of binding of approximately -600 cal/mol and a binding affinity of approximately -1.25 x 10(2) M-1. Finally, the rate of degradation of deslorelin by alpha-chymotrypsin was decreased by 33% in the presence of HPbetaCD.. These results indicate that there is an interaction between HPbetaCD and deslorelin, which involves the inclusion of aromatic amino acids of deslorelin into the hydrophobic cavity of the cyclodextrin. This inclusion, providing steric hindrance, may be one of the mechanisms by which HPbetaCD reduces enzymatic hydrolysis of deslorelin.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; alpha-Cyclodextrins; Amino Acids, Aromatic; beta-Cyclodextrins; Chymotrypsin; Cyclodextrins; Drug Interactions; Drug Stability; Excipients; Gonadotropin-Releasing Hormone; Protein Conformation; Spectrometry, Fluorescence; Thermodynamics; Triptorelin Pamoate