alpha-chymotrypsin and betadex

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

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 relative effectiveness of two beta-cyclodextrin derivatives, i.e., dimethyl-beta-cyclodextrin (DM beta CD) and hydroxypropyl-beta-cyclodextrin (HP beta CD), in enhancing enteral absorption of insulin was evaluated in the lower jejunal/upper ileal segments of the rat by means of an in situ closed loop method. The incorporation of 10% (w/v) DM beta CD to a 0.5 mg/ml porcine-zinc insulin solution dramatically increased insulin bioavailability from a negligible value (approximately 0.06%) to 5.63%, when administered enterally at a dose of 20 U/kg. However, addition of 10% (w/v) HP beta CD did not improve enteral insulin uptake significantly with a bioavailability of only 0.07%. Similarly, the pharmacodynamic relative efficacy values obtained after the enteral administration of 20 U/kg insulin, 20 U/kg insulin with 10% HP beta CD, and 20 U/kg insulin with 10% DM beta CD were 0.24%, 0.26%, and 1.75%, respectively. Biodegradation studies of 0.5 mg/ml insulin hexamers by 0.5 microM alpha-chymotrypsin revealed no inhibitory effect on the enzymatic activity by the two cyclodextrins. On the contrary, the apparent first-order rate constant increased significantly in the presence of 10% DM beta CD, suggesting insulin oligomer dissociation by DM beta CD. Histopathological examination of the rat intestine was performed to detect tissue damage following enteral administration of the beta-cyclodextrin derivatives. Light microscopic inspection indicated no observable tissue damage, thereby arguing direct membrane fluidization as the primary mechanism for enhanced insulin uptake. This study indicates the feasibility of using cyclodextrins as mucosal absorption promoters of proteins and peptide drugs.

Topics: Administration, Oral; Animals; beta-Cyclodextrins; Biological Availability; Blood Glucose; Chromatography, High Pressure Liquid; Chymotrypsin; Cyclodextrins; Injections, Intravenous; Insulin; Intestinal Absorption; Intestinal Mucosa; Male; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Swine

The water-insoluble aluminium salt of beta-cyclodextrin sulphate (Al.beta-CyD-Sul) was used as a stabilizer and sustained-release carrier for recombinant human basic fibroblast growth factor (bFGF). An adsorbate of bFGF with Al.beta-CyD-Sul was prepared by incubating the protein with a suspension of Al.beta-CyD-Sul in water. The mitogenic activity of bFGF released from the adsorbate, as indicated by the proliferation of kidney cells of baby hamster (BHK-21), was almost comparable with that of the intact bFGF. Al.beta-CyD-Sul significantly protected bFGF from proteolytic degradation by pepsin and alpha-chymotrypsin, compared with the water-soluble sodium salt. The in-vitro release of bFGF from the adsorbate was sustained in proportion to a rise in the ratio of Al.beta-CyD-Sul to the protein in the adsorbate. Of the bFGF preparations evaluated, the adsorbate of bFGF with Al.beta-CyD-Sul, when given subcutaneously to the rat, showed the most prominent increase in the formation of granulation tissues, due to the stabilization and slow-release of the mitogen. The limited data presented here suggest that the adsorbate of bFGF with Al.beta-CyD-Sul has a potent therapeutic efficacy for wound healing, and may be applicable to oral protein formulations for the treatment of intestinal mucosal erosions.

Topics: Animals; beta-Cyclodextrins; Cell Line; Chymotrypsin; Cricetinae; Cyclodextrins; Delayed-Action Preparations; Excipients; Fibroblast Growth Factor 2; Humans; Mitogens; Pepsin A; Rats; Recombinant Proteins; Spectrophotometry, Ultraviolet; Wound Healing

The synthesis is described of a derivative of cyclomaltoheptaose (beta-cyclodextrin) to which the tripeptide Ser-His-Asp, the catalytic triad found in chymotrypsin, has been coupled. The derivative enhanced the rates of hydrolysis of activated esters, as measured by the release of p-nitrophenol, and the formation of amine bonds.

Topics: Amides; Amino Acid Sequence; beta-Cyclodextrins; Carboxylic Acids; Chemical Phenomena; Chemistry; Chymotrypsin; Cyclodextrins; Dextrins; Esters; Hydrolysis; Nitrophenols; Oligopeptides; Starch

The thermal and pH stability of "beta-benzyme", an artificial chymotrypsin based on beta-cyclodextrin, has been studied and compared with the stability of real chymotrypsin. Artificial chymotrypsin is vastly superior to real chymotrypsin with regard to both temperature and pH stability. The reasons for this increased stability are discussed.

Topics: beta-Cyclodextrins; Chymotrypsin; Cyclodextrins; Dextrins; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Starch; Thermodynamics