Glycosyl part identified within Balanites aegyptiaca fruit protease.

The many milk-clotting proteases from plant are glycosylated; attachment of monosaccharides to enzyme is an advantage for its activity and stability. In this study, gas chromatography coupled to mass spectrometry-electrospray ionization was used to identify glycans bond to proteases purified from Balanites aegyptiaca fruits pulp through cation exchange chromatography. Carbohydrates were identified according to the retention time and the ion at m/z after derivation by heptafluorobutyric acid. The chromatograms obtained from monosaccharides analysis revealed the presence of galactose, mannose, arabinose, xylose, rhamnose and glucuronic acid. The mass spectrometry-electrospray ionization spectra corroborated these findings.

Thermal denaturation and gelation of rubisco: effects of pH and ions.

In order to understand the mechanism of thermal gelation of rubisco, its native and heat denatured states were characterized by absorbance, fluorescence and circular dichroïsm spectroscopies as well as by differential scanning calorimetry in the presence of various salts. It appears that during the denaturation process, divalent anions are released while divalent cations are fixed by the protein, while it is disorganized and while the environment of its aromatic chromophores becomes more hydrophilic. The pH transition of gelation is shifted 1-2 pH units higher than the transition of denaturation temperature which occurs near the isoelectric point of the native molecule. This shift probably corresponds to the breaking of saline bridges within the protein molecule. Finally, a large effect of divalent cations on the phase diagram indicates that a particular denatured state is attained when these cations are in the denaturation medium.

Differential scanning calorimetric studies of the effects of ions and pH on ribulose 1,5-bisphosphate carboxylase/oxygenase.

Lucerne rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase, EC 4.1.1.39) was purified by ammonium sulfate fractionation and gel chromatography. Differential scanning calorimetry (d.s.c.) was used to study the effects of pH in the range 3.5 to 11.4, and the effects, at pH 7.5, of various salts at ionic strength lower than 0.3 (NaH2PO4, (NH4)2SO4, Na2SO4, NaCl, MgCl2, CaCl2) on the thermal denaturation of the protein. Van't Hoff and calorimetric enthalpies, and the change in heat capacity between the native and denatured states were calculated from the experimental data. The effects of salts on the thermal denaturation seem to follow the Hofmeister series. In some cases, the thermal denaturation may be interpreted as a two-step transition of the polypeptide chains. Moreover, comparison of the results with literature data suggests that the thermal denaturation parameters depend on the botanical origin of rubisco and are affected by the conditions of its purification.