High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

Characterization of antibodies to chicken riboflavin carrier protein: Antigenicity of the tryptic fragments

The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.