muramidase and sodium-perchlorate

Affinity chromatographic assays for thrombin were developed using two aptamers as affinity ligands. The efficient capture and step elution of thrombin with NaClO4 enabled the determination of thrombin by using either absorbance or fluorescence detection. Preconcentration of thrombin on the affinity column improved the detection limit of thrombin to 0.1 nM. Using an aptamer for the fibrinogen-binding site of thrombin and a second aptamer for the heparin-binding site, a sandwich chromatographic assay was developed, showing improved selectivity of thrombin detection and eliminating the need for labeling thrombin in the sample. The increased local concentration of aptamers immobilized on monolithic columns favored the formation of aptamer-thrombin complexes, resulting in improved retention and detection of thrombin at trace levels.

Topics: Animals; Aptamers, Nucleotide; Binding Sites; Chromatography, Affinity; Fibrinogen; Humans; Immunoglobulin G; Muramidase; Perchlorates; Reproducibility of Results; Serum Albumin; Sodium Compounds; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thrombin; Transferrin

Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [(35)S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [(35)S]sulfate in the control, 80% of the [(35)S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH(4)Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells.

Topics: Ammonium Chloride; Chondroitin ABC Lyase; Chondroitin Sulfates; Chromatography, Affinity; Enzymes, Immobilized; Humans; Kinetics; Lysosomes; Mannosephosphates; Muramidase; Perchlorates; Protein Binding; Protein Transport; Proteoglycans; Sodium Compounds; Sulfates; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; U937 Cells

Aqueous two-phase systems composed of ethylene oxide/propylene oxide random co-polymers, EO30/PO70 or Ucon (EO50/PO50), in the top phase and dextran T500 in the bottom phase, have been studied. The cloud point diagram for EO30/PO70 in water solution was determined. EO30/PO70 has a cloud point of 32 degrees C at a concentration of 10% (w/w). The phase diagram for the system EO30/PO70-dextran T500-water was determined. Salt effects have been studied on the partitioning of two model proteins, bovine serum albumin and hen egg white lysozyme, in EO30/PO70-dextran and Ucon-dextran systems. Ions with different hydrophobicity, i.e., with different position in the Hofmeister or lyotropic series, were investigated with reference to their effect on protein partition. The counterion hydrophobicity was shown to have a strong influence on the partitioning of BSA and lysozyme. Most extreme partitioning was obtained with hydrophobic (chaotropic) ions like CIO4- and I-. A comparison of protein partitioning between PEG-dextran and EO30/PO70-dextran has been done. A more extreme protein partitioning was obtained in the EO30/PO70-dextran containing system. Temperature-induced phase separation was studied with EO30/PO70 at 45 degrees C. Both BSA and lysozyme were completely partitioned to the water phase formed above the cloud point of EO30/PO70. Model calculations, based on Flory-Huggins theory of polymer solutions, have been done which could reproduce the salt effect on the protein partitioning in aqueous-two phase system.

Topics: Bromides; Chemical Phenomena; Chemistry, Physical; Glycine; Ions; Models, Chemical; Muramidase; Perchlorates; Polyethylenes; Polypropylenes; Serum Albumin, Bovine; Sodium Chloride; Sodium Compounds; Sodium Iodide; Water