TSK-GEL Column Research Articles

Non-leaky vesiculation of large unilamellar vesicles (LUV) induced by plasma high density lipoproteins (HDL): Detection by HPLC

Interaction of large unilamellar phosphatidylcholine vesicles (LUV, 75nm) and plasma high density lipoproteins (HDL) resulted in a non-leaky vesiculation of LUV. This vesiculation was detected by a HPLC-system consisting of a combination of three TSK-gel columns (6000PW, 5000PW, 3000SW). With increasing incubation time liposomal [14C]PC, entrapped [3H]inulin, and apoprotein of HDL origin decreased. The decrease was accompanied by a formation of new particles, consisting of liposomal PC and apoprotein. These particles also enclosed [3H]inulin, reflecting a hydrophilic inner space. The formation of the particles reached a maximum after one day of incubation. Retention time was 21 minutes for LUV, 28 minutes for the new particles, and 36 minutes for HDL. In vesicles with membranes consisting of phosphatidylcholine and 30% cholesterol no interactions were observed.

Rapid and simple isolation procedure for three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex.

A novel procedure was developed for rapid separation of the three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex by high performance liquid chromatography on a gel filtration column. The complex was dissociated and separated into two fractions of the first dihydrolipoamide succinyltransferase and a second yellow fraction within 1 h by chromatography on a preparative TSK-GEL G4000SW column equilibrated with 0.05 M potassium phosphate buffer (pH 7.0) containing 0.7 M guanidine hydrochloride, 0.05% Triton X-100 and 2 mM dithiothreitol at 10 degrees C. The dihydrolipoamide succinyltransferase fraction was further purified by incubation with 0.5% sodium deoxycholate and subsequent ammonium sulfate fractionation. The other two component enzymes, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase were separated from the second yellow fraction by chromatography on a calcium phosphate gel-cellulose column. The TSK-GEL column permitted very rapid dissociation and separation of the three component enzymes accompanied by good preservation of their activities and high overall yields.

A regulatory protein for orthophosphate-regulated cyclic phosphodiesterase in [formula omitted

A regulatory protein for orthophosphate-regulated cyclic phosphodiesterase (cPDase) was detected in mycelial extracts of Neurospora crassa . The protein, designated neucrassin, was precipitated by ammonium sulfate between 60 to 100 % saturation, and fractionated by gel filtration through a TSK-gel column. The molecular weight was estimated to be 65,000. Neucrassin inhibited the hydrolyzing activity of cPDase for cyclic 3′,5′-AMP in the presence of MnCl 2 in a noncompetitive manner, whereas it stimulated the activity for cyclic 2′,3′-AMP over a wide ranges of pH's, between 2.8 to 6.8.

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10 4 and 10 5. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.

High-Performance Gel-Permeation Chromatography of Bovine Skim Milk Proteins

Abstract The separation of bovine skim milk proteins by gel-permeation high performance liquid chromatography was examined. Toya-Soda TSK-GEL (Type SW) columns were used with an eluent of .05 M phosphate buffer (pH 6.80) containing .1 M sodium sulfate at .5 ml/min. Bovine whole milk was centrifuged to remove lipids, and the resultant skim milk directly injected. A 2000SW column yielded three protein peaks: 1 = casein, IgG and BSA; 2 = 6-lactoglobulins and BSA; and 3 = α-lactalbumin and BSA. A 3000SW plus 2000SW column system with a 30 μl injection volume yielded four protein peaks: 1 = minor amounts of α - and β-casein; 2 = casein, BSA and IgG; 3 = β-lactoglobulins; and 4 = α1-lactalbumin. A 3000SW plus 2000SW column system with a 10 μl injection volume yielded five protein peaks: 1 = casein; 2 = IgG; 3 = BSA; 4 = β-lactoglobulins; and 5 = α-lactalbumin. Both the single column and dual column applications yielded three nonprotein peaks, which were dialyzed from solution. Thus, a high speed analytical separation of milk proteins was achieved according to molecular size, but this application is highly dependent on sample size.