Renate Bublitz

Composition analysis of detergents of the polyoxyethylene type: comparison of thin-layer chromatography, reversed-phase chromatography and matrix-assisted laser desorption / ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can be used to determine the distribution of single polymer species of non-ionic detergents of the polyoxyethylene type (Triton X-100 and 114, Tween 20 and Brij 35). Thin-layer chromatographic (TLC) and reversed-phase chromatographic (RPC) methods are presented which may separate single polymer species as verified by MALDI-MS. Comparison of chromatographic and MALDI-MS data show Poisson-like distributions for Triton X-100 without marked differences between different methods. Distribution parameters obtained with Triton X-100 charges from different suppliers are very similar. The RPC method used can be scaled up for preparation of pure detergent species.

Glycosylphosphatidylinositol-Specific Phospholipase D of Human Serum Activity Modulation by Naturally Occurring Amphiphiles

Abstract The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6000-fold purified enzyme. This was obtained in 100 μg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) cochromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.

Gel chromatographic characterization of the hydrophobic interaction of glycosylphosphatidylinositol-alkaline phosphatase with detergents.

Abstract The interaction of a glycosylphosphatidylinositol (GPI) protein with different detergents was studied for the first time with a purified protein. Four differently hydrophobic fractions of GPIalkaline phosphatase (GPIAP) from calf intestine were used as model proteins. The mode of interaction was determined by investigating (i) the selfaggregation behaviour of the GPIAP fractions, (ii) the interference of detergents with GPIAP binding to octylSepharose, and (iii) the elution of GPIAP bound to octylSepharose. It was shown that polyoxyethylenetype detergents surprisingly interact much stronger than noctylglucoside with GPIAP, which is in contrast to the known behaviour of GPIproteins in natural membranes. Gel filtration chromatography of Triton X-100 at concentrations above the critical micellar concentration yields three different micelle species with apparent molecular weights of about 166, 54, and 16 kDa. GPIAP fraction II, which is shown to bear only one anchor per dimer, does not bind to any of these micelles. We demonstrate that a complex is formed containing about 150 Triton X-100 molecules and about 4700 molecules of water per molecule of GPIAP dimer. The experimental findings are in accordance with a simple geometrical model based on the physical data of fatty acids and the arrangement, mean size, and shape of Triton X-100 molecules.