raffinose and alpha-chymotrypsin

An acidic α-galactosidase designated as PDGI (Pleurotus djamor α-galactosidase) was purified to homogeneity with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 53.5°C, respectively. It displayed great pH stability within the pH range 3.0-10.0. Besides, the enzyme harbored remarkable resistance to acid protease and varying degrees of tolerance to other proteases: trypsin>collagenase Type-I>α-chymotrypsin neutral protease>proteinaseK. It was strongly inhibited by K

Topics: alpha-Galactosidase; Amino Acid Sequence; Chymotrypsin; Enzyme Stability; Fungal Proteins; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Metals; Molecular Weight; Oligosaccharides; Pleurotus; Raffinose; Substrate Specificity

A reported discrepancy between quantitative estimates of the extent of enhanced alpha-chymotrypsin dimerization in the presence of sucrose is traced to different consequences of using an incorrect value of the buoyant molecular weight in the analysis of sedimentation equilibrium distributions. Support is thereby provided for the earlier contention that the effect of sucrose, as well as of glucose and raffinose, on dimerization may be rationalized quantitatively in terms of molecular crowding by an inert cosolute.

Topics: Chymotrypsin; Dimerization; Glucose; Mathematics; Molecular Weight; Raffinose; Sucrose; Thermodynamics; Ultracentrifugation

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

Topics: Chymotrypsin; Dimerization; Energy Transfer; Glucose; Kinetics; Protein Binding; Raffinose; Sucrose