A partially purified phosphopeptide fraction with low N/P ratio has been obtained from the small intestinal contents of rats, which was collected at the first one hour after 1.5hr spaced-feeding of the diet containing 20%, casein. After precipitation of protein with trichloroacetic acid, acid-soluble fraction was chro matographed on the column of Sephadex G-25. A fraction containing large peptide(s) having the N/P atomic ratio of about 7 to 9 and free from lower molecular substances was rechromatographed with Sephadex G-50. This fraction, amounted nearly 30% of the total non-protein nitrogen of the intestinal contents, showed high calcium-binding activity as determined with Chelex-100. These results suggest a special role of phosphopeptide stage on the stimulating calcium absorption in the intestinal tract during the digestion of casein.
A chromatographic determination method was developed for the absolute configuration of an acyclic secondary alcohol using the characteristic functions of 1,5-difluoro-2,4-dinitrobenzene (FFDNB). This method relies on the formation of the fixed favorable conformation of a secondary alcohol-DLA (2,4-dinitrophenyl-5-leucinamide) derivative and its recognition by ODS silica gel. The secondary alcohol reacted first with FFDNB under mild basic conditions, and L-leucinamide or DL-leucinamide was then introduced into the secondary alcohol-FDNB derivative. Because the conformations of the resulting alcohol-DLA derivatives were rigidly fixed by the dinitrobenzene plane, the absolute configuration at the asymmetric carbon of the secondary alcohol tested can be definitely deduced by the elution behavior of both of the diastereomers in the HPLC and/or LC/MS. One of the diastereomers has the cis (Z)-type arrangement of two more hydrophobic substituents of the alcohol and leucinamide moieties, the more hydrophobic side chain and isobutyl groups, to the plane of the dinitrobenzene, whereas another diastereomer has the opposite arrangement (trans (E)-type). Therefore, the cis arrangement interacts more strongly with the ODS silica gel and has a longer retention time than that of the trans-type arrangement. This established method was successfully applied to various chiral acyclic secondary alcohols including chloramphenicol and 4-hydroxyphenyllactic acid. Finally, the limitations of this method were also examined.
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