Active Transport of Alcohol in Corynebacterium acetophilum

The transport of alcohols was studied in Corynebacterium acetophilum, which was isolated as a strain growing well on acetate and ethanol. The transport of ethanol was found to be inducible by ethanol, n-propanol, n-butanol, and acetate, whereas transport of methanol occurred by noninducible passive diffusion. The entry of ethanol into the cells occurred against a concentration gradient and showed saturation kinetics with two K(m) values of 2.4 x 10(-5) M and 6.0 x 10(-5) M. Uptake of ethanol was inhibited by sodium azide, sodium cyanide, 2,4-dinitrophenol, and p-chloromercuribenzoate. The transport of ethanol was competitively inhibited by normal alcohols, but not by iso- or tert-alcohols. From these studies, we concluded that an inducible active alcohol transport system mediates the entry of ethanol, n-propanol, or n-butanol into the cells of C. acetophilum.     

Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n- propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.     

Solvent isotope effect on thermodynamics of hydration

Partial molar heat capacities of five linear alcohols (methanol, ethanol, n-propanol, n-butanol, n-pentanol) and five N-substituted amides (n-propionamide, N-methylformamide, N-methylacetamide, N-methylpropionamide, N-ethylacetamide) in aqueous D(2)O solution have been measured at 25 degrees C. The heat capacities of transfer of these compounds from H(2)O to D(2)O were calculated using previously reported (Makhatadze et al., Biophys. Chem. 64 (1997) 93) values of partial heat capacities of alcohols and amides in aqueous H(2)O solutions. It is shown that the sign and magnitude of the heat capacity change upon transfer from H(2)O to D(2)O depends on the relative amount of polar and non-polar solvent accessible surface areas of solute. Analysis shows that transfer of non-polar surface from H(2)O to D(2)O is accompanied by a positive heat capacity change. In contrast, transfer of polar surface from H(2)O to D(2)O occurs with negative heat capacity change. Estimates show that the solvent isotope effect on the heat capacity changes upon protein unfolding can be predicted using the changes of the polar and non-polar surface area changes upon protein unfolding and the transfer data of model compounds. Analysis of the thermodynamic functions of transfer of non-polar compounds from H(2)O to D(2)O shows puzzling behavior which contradicts current definitions of the hydrophobic effect.     

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.     

A novel type of formaldehyde-oxidizing enzyme from the membrane of Acetobacter sp. SKU 14

Membrane-bound NADP-independent formaldehyde-oxidizing enzyme was purified to homogeneity from the membrane of Acetobacter sp. SKU 14 isolated in Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Tween 20 at pH 2.85, and purified to homogeneity through the steps of column chromatographies on DEAE-Sephadex A-50 and Q-Sepharose in the presence of 0.1% Tween 20 and 0.1% Triton X-100. The enzyme purified together with a cytochrome c showed a single protein band on native-PAGE, and was dissociated into three different subunits upon SDS-PAGE with molecular masses of 78 kDa, 55 kDa, and 18 kDa. The purified enzyme was finally characterized as a quinoprotein alcohol dehydrogenase (QADH), and this is the first indication that QADH highly oxidizes formaldehyde. The substrate specificity of the enzyme was found to be broad toward aldehydes and alcohols, and alcohols, especially n-butanol, n-propanol, and ethanol, were oxidized more rapidly than formaldehyde.     

Azotobacter vinelandii aldehyde dehydrogenase regulated by sigma(54): role in alcohol catabolism and encystment

Encystment in Azotobacter vinelandii is induced by n-butanol or beta-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named aldA. An aldA mutation impaired bacterial growth on n-butanol, ethanol, or hexanol as the sole carbon source. Expression of aldA increased in cells shifted from sucrose to n-butanol and was shown to be dependent on the alternative sigma(54) factor. A mutation in rpoN encoding the sigma(54) factor also impaired growth on alcohols. Encystment on n-butanol, but not on BHB, was impaired in aldA or rpoN mutants, indicating that n-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment.     

Effect of alcohols on aqueous lysozyme-lysozyme interactions from static light-scattering measurements

Alcohols have been widely used as protein denaturants, precipitants and crystallization reagents. We have studied the effect of alcohols on aqueous hen-egg lysozyme self-interactions by measuring the osmotic second virial coefficient (B22) using static light scattering. Addition of alcohols increases B22, indicating stronger protein-protein repulsion or weaker attraction. For the monohydric alcohols used in this study (methanol, ethanol, 1-propanol, n-butanol, iso-butanol and trifluoroethanol), B22 for lysozyme reaches a common plateau at approximately 5% (v/v) alcohol, while glycerol increases B22 more than monohydric alcohols. For a 0.05 M NaCl hen-egg lysozyme solution at pH 7, B22 increases from 2.4 x 10(-4) to 4.7 x 10(-4) ml mol/g2 upon addition of monohydric alcohols and to 5.8 x 10(-4) ml mol/g2 upon addition of glycerol. We describe the alcohol effect using a simple model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions. In this model, the increased lysozyme repulsive forces in the presence of monohydric alcohols are interpreted in terms of adsorption of alcohol molecules on hydrophobic sites on the protein surface. This adsorption reduces attractive hydrophobic protein-protein interactions. A thicker lysozyme hydration layer in aqueous glycerol solution can explain the glycerol-increased lysozyme-lysozyme repulsion.     

Changes of Acetylcholinesterase Activity in Brain Areas and Liver of Sucrose- and Ethanol-Fed Rats

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.     

Aggregation of wool keratin intermediate filament proteins

The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.     

Direct enantiomeric resolutions of chiral triazole pesticides by high-performance liquid chromatography

Cellulose-tris (3,5-dimethylphenylcarbamate; CDMPC) was synthesized and coated on aminopropylsilica to prepare chiral stationary phase (CSP). Normal-phase high-performance liquid chromatography (HPLC) methods for the resolutions of five chiral triazole pesticides, diniconazole, tebuconazole, hexaconazole, triadimefon and flutriafol, on the CSP were developed. Several operating parameters such as mobile phase composition, modifier and column temperature were studied for the optimization of the resolutions. Better separations were achieved using 2% iso-butanol for diniconazole, 2% ethanol for tebuconazole, 2% iso-propanol for hexaconazole, 1% n-butanol for triadimefon and 2% n-propanol for flutriafol as modifiers in n-hexane at 0 degrees C with the resolution factors (Rs) of 1.62, 1.66, 2.46, 1.68 and 1.98, respectively. Low temperature was better for the resolutions. Validation of the methods included linearity and precision.     

Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents

Hydrophobic silica aerogels modified with methyl group were applied as support to immobilize Candida rugosa lipase (CRL). At the adsorption process, different alcohols were used to intensify the immobilization of CRL. The results showed that n-butanol wetting the hydrophobic support prior to contacting with enzyme solution could promote lipase activity, but the adsorption quantity onto the support decreased. Based on this, a novel immobilization method was proposed: the support contacted with enzyme solution without any alcohols, and then the immobilized enzymes were activated by 90% (V) n-butanol solution. The experimental results showed that this method could keep high adsorption quantity (413.0 mg protein/g support) and increase the lipase specific activity by more than 50%. To improve the stability of immobilized lipase, the support after adsorption was contacted with n-octane to form an oil layer covering the immobilized lipases, thus the leakage can be decreased from over 30–4% within 24 h. By utilizing proper cosolvents, a high enzyme activity and loading capacity as well as little loss of lipase was achieved without covalent linkage between the lipase and the support. This is known to be an excellent result for immobilization achieved by physical adsorption only.     

Pseudomonas cepacia lipase--mediated transesterification reactions of hydrocinnamates

Use of lipase from Pseudomonas cepacia in transesterifcation reactions of ethyl hydrocinnamate with different alcohols has been examined. Among the alcohols tested, viz., n-butanol, iso-amyl alcohol, benzyl alcohol, n-octanol and 1-phenylethanol, only n-butanol yielded the transesterified product. Among the solvents tested, viz., n-heptane, n-hexane, cyclohexane, toluene, diisopropylether and n-butanol, the initial rate of transesterification proceeded in the order cyclohexane > n-heptane > n-hexane > diisopropylether > n-butanol > toluene. Using hexane as the solvent and a substrate to enzyme ratio of 1:5, the substrate to alcohol ratio was varied to maximize the yield. n-Butyl hydrocinnamate was obtained in 92% yield in 48 hr by employing a 1:1 (wt/wt) ratio of ethyl hydrocinnamate to lipase and a 1:5 (vol/vol) ratio of ethyl hydrocinnamate to n-butanol in hexane.     

A new alkaline elastase of an alkalophilic bacillus

A new alkaline elastase was purified from the culture broth of an alkalophilic Bacillus sp. Ya-B. This was a serine proteinase. Molecular weight was 25,000. The optimum pH for elastin and casein was 11.75. The enzyme had very high specific activity, 12,400 units/mg protein for casein, and 2,440 units/mg protein for elastin at the optimum pH. It showed marked preference for elastin. The relative activity of elastin/casein of this enzyme was 17 and 6 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. This enzyme also had higher keratin and collagen hydrolyzing activity in comparison with subtilisin.     

Triphasic effects of short chain n-alcohols on synaptic membrane transport of choline and of gamma-aminobutyric acid

n-Alcohols, when added in increasing concentrations, had an unusual triphasic effect on the uptake of choline and of gamma-aminobutyric acid by isolated synaptosomes. There was slight inhibition of these uptakes at low n-alcohol concentrations, followed by a sharp peak of uptake enhancement, and then greater inhibition. The n-alcohol concentrations required for these effects were proportional to published n-alcohol membrane/buffer partition coefficients, with the peaks of uptake enhancement occurring at 60 mM n-propanol, 20 mM n-butanol and 7.5 mM n-pentanol. Synaptosomal membrane potential, as estimated from synaptosomal accumulation of the permeant cation [3H]tetraphenylphosphonium, was not affected by n-alcohols in the concentrations used in this study, suggesting that neither the inhibitory or enhancing effects of these n-alcohols were attributable to changes in trans-synaptosomal membrane ion gradients. The inhibiting and enhancing effects of n-alcohols could be reproduced in determinations of gamma-aminobutyric acid uptake by isolated synaptic plasma membranes, suggesting that the observed effects are due to a direct action of the n-alcohols on the synaptosomal plasma membrane. These effects may be attributable to a change in membrane binding of these alcohols from the membrane core to the membrane surface as alcohol concentration is increased.     

Reversible and irreversible interactions between elastin and plasma lipoproteins

The interactions between radiolabeled, human plasma lipoproteins and elastin derived from bovine ligamentum nuchae were investigated using a washout technique. The interaction was characterised by Ki, a coefficient of irreversible binding, and Kr, the reversible partition coefficient. For both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) the Ki values decreased as total lipoprotein concentration increased, suggesting that the binding is saturable, and were similar in magnitude to those measured by other workers using elastin derived from the human aorta. For both LDL and HDL the Kr values were independent of lipoprotein concentration in the range 0.1 microgram/ml-1.5 micrograms/ml. At a total protein concentration of 1.5 mg/ml in the incubation medium, the reversible interactions were comparable in magnitude to the irreversible.     

Interaction between human cathepsins K, L, and S and elastins: mechanism of elastinolysis and inhibition by macromolecular inhibitors

Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins.     

Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis

The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results from the protein quantification. After desorption, the IgG gamma-chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.