Gerhard A. Cumme

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.

An Improved Method for Checking HTS/uHTS Liquid-Handling Systems

An efficient method is presented to determine precision and accuracy of multichannel liquid-handling systems under conditions near to application. Themethod consists of gravimetrical determination of accuracy and optical determination of precision based on the dilution of absorbing and fluorescent dye solutions in microplates. Mean delivery volume per well can be determined with precision better than a 0.04% coefficient of variation (CV). Optical signal precision, CV(S), is improved by multiwavelength measurements. Precision of absorbance measurement yields a better resolution than precision of fluorescence measurement (0.3% and 1.5%, respectively), indicating that absorbance measurements should be preferred. From CV(S), an upper bound of the precision of the volumes delivered is derived. Method performance is demonstrated with the dispenser CyBi™-Drop and the pipettor CyBi™-Well using different ejection principles; with commonly used fluids; with 96-, 384-, and 1536-well microplates; and with photometric and fluorometric indicators. Precision of the volumes delivered, as obtained with optimized methods, all plate formats, and both devices, is better than 2% CV with 2 µ L set volume and about 1% CV with higher set volumes.

UV measurements in microplates suitable for high-throughput protein determination.

An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.6% only with the Path Check option of the reader, protein concentration can be determined without any individual calibration. Samples in the volume range of 60-250 microl can be analyzed without time-consuming and expensive treatment and without sample loss. Using a special 96 or 384 parallel dialyzing device, low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents, can effectively be removed. Application examples for serum protein separation are also shown in the presence of the strongly UV absorbing detergent Triton X-100.

Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation.

Biomarker search using multidimensional native liquid fractionation of serum in microplates was evaluated. From different donors, homologous sample fractions with UV absorbance depending on state of illness were selected, and their constituents were identified and quantitated by MS. Analysis of sera of patients with Alport syndrome and severe inflammation proved the reliability of the method by confirming characteristic alterations. Moreover, 23 new marker candidates were detected for Alport syndrome, some of them being involved in matrix degradation and repair, and 33 new candidates for severe inflammation, among them alpha1B-glycoprotein cysteine-rich secretory protein and an apparently low molecular-weight albumin variant.

Determination, purification, and characterization of alpha-NADH and alpha-NADPH.

Publisher Summary This chapter describes the two methods for the quantitative determination of small amounts of α-anomers of nicotinamide adenine dinucleotide (NAD), NADH, nicotinamide adenine dinucleotide phosphate (NADP), and NADPH in the presence of large amounts of β-anomers. These methods use ion-exchange chromatography or reversed-phase chromatography combined with enzymatic oxidation of β-NADH and β-NADPH by lactate dehydrogenase (LDH) and glutathione reductase, respectively. They are used to determine the rate and equilibrium constants of the α–β anomerization reactions of NADH and NADPH. Because commercially available α-NADH preparations are not pure, a method for the preparation of 20- to 50-mg amounts of α-NADH with purity better than 96% is presented and spectrophotometric and fluorometric properties of these preparations are investigated. The spontaneous α-to-β and β-to-α anomerization reaction of NADH is investigated quantitatively by following the α-to-β reaction either by measuring the residual absorption or by using the relatively high specificity of dehydrogenases for β-NADH, directly measuring anomerization velocity in the presence of large amounts of dehydrogenases.

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.

Determination of rate constants of monoclonal antibodies to enzymes

A novel graphical method for determining rate constants of the immune reaction of enzyme-inhibiting or -activating antibodies has been evaluated. The experimental determination of kinetic constants does not require purification of antibody and enzyme nor any separation step of bound and free entities. The resultant enzyme activity is used as a measurement of the extent of enzyme-antibody complex formation. The plot ln magnitude of v - v infinity versus t (v = enzyme activity at time t, v infinity = enzyme activity at infinite time) yields straight lines in the case of antibody excess. Slope and vertical intercept of that primary plot can be used for secondary plots to obtain association (k1) and dissociation (k-1) rate constant, the dissociation constant KD of the complex and the residual enzyme activity of the complex (g/f). The suitability of the graphical method has been established experimentally using the mab IB 10B8 (2) which inhibits alkaline phosphatase activity. With the homogeneous assay in the presence of substrate as well as a microassay in the absence of substrate, k1 and g/f were found to be 4.68 x 10(7) M-1 min-1; 3.74 x 10(7) M-1 min-1 and 0.035; 0.107 respectively. For k-1 and KD, only crude estimates could be derived. The method has been tested for model discrimination using computer simulated data.

Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [P] = a + b x t - c x exp(-d x t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities. (i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25 degrees C to 37 degrees C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (E) and substrate-bound (ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher Km value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II-V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II-V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.

Steady state and quasi-equilibrium enzyme models: a comparison of equation types

Abstract The velocity equations of steady state and quasi-equilibrium models are proved to have the following equivalence properties: (1) For a large class of steady state enzyme models there exist quasi-equilibrium models with identical velocity equations. (2) To any steady state enzyme model there corresponds a quasi-equilibrium model such that their velocities do not differ measurably from each other if the effector concentrations are not exactly equal to zero and if only insignificant portions of the effectors are bound to the enzyme. Thus even if the true type of an enzyme mechanism is not known, its kinetic properties may well be adequately described by quasi-equilibrium velocity equations under such conditions. Therefore, if no more is sought than an empirical velocity equation (e.g., for mathematical description of an enzyme within a metabolic system), then the mathematically much simpler quasi-equilibrium models may well be used to facilitate finding an equation which describes the observed kinetic properties.