Werner Schröder

Analysis of plasma protein adsorption on polymeric nanoparticles with different surface characteristics

Plasma protein adsorption patterns on colloidal drug carriers acquired after i.v. administration depend on their surface characteristics and are regarded as key factors for their in vivo organ distribution. Polymeric latex particles with strongly differing surface properties were synthesized as models for colloidal drug carriers for tissue-specific drug targeting via the intravenous route. Physicochemical characterization was performed for size, surface charge density, zeta potential, and surface hydrophobicity. The interactions with human plasma proteins were studied by way of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Considerable differences in protein adsorption on the latex particles were detected with regard to the total amount of surface-bound protein on the various particle types as well as specific proteins adsorbed, for example, fibrinogen, albumin, and a recently identified plasma glycoprotein. Possible correlations between protein adsorption patterns and the physicochemical characteristics and topography of the polymeric surfaces are shown and discussed. Knowledge about protein-nanoparticle interactions can be utilized for the rational design of colloidal drug carriers and also may be useful for optimizing implants and medical devices.

The role of plasma proteins in brain targeting: species dependent protein adsorption patterns on brain-specific lipid drug conjugate (LDC) nanoparticles.

The in vivo organ distribution of particulate drug carriers is decisively influenced by the interaction with plasma proteins after i.v. administration. Serum protein adsorption on lipid drug conjugate nanoparticles, a new carrier system for i.v. application, was investigated by 2-dimensional electrophoresis (2-DE). The particles were surface-modified to target them to the brain. To assess the protein adsorption pattern after i.v. injection in mice prior to in vivo studies, the particles were incubated in mouse serum. Incubation in human serum was carried out in parallel to investigate similarities or differences in the protein patterns obtained from men and mice. Distinct differences were found. Particles incubated in human serum showed preferential adsorption of apolipoproteins A-I, A-IV and E. Previously, preferential adsorption of ApoE was reported as one important factor for targeting of Tween(R)80 modified polybutylcyanoacrylate nanoparticles to the brain. Preferential adsorption of ApoA-I and A-IV took place after incubation in mouse serum, adsorption of ApoE could not be clearly confirmed. In vivo localization of the LDC nanoparticles at the blood-brain barrier and diffusion of the marker Nile Red into the brain could be shown by confocal laser-scanning microscopy. Differences of the obtained adsorption patterns are discussed with regard to their relevance for correlations of in vitro and in vivo data obtained from different species.

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)-acycloguanosine inhibitor

Calf spleen purine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)‐acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three‐fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 Å to an R‐factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of nucleosides.

Isolation and partial characterization of the major protein of the outer membrane of Pasteurella haemolytica and Pasteurella multocida

The N-terminal amino acid sequence of the 35 kDa (p35) major outer membrane protein (MOMP) of P. multocida shared a strong homology with those of homotrimeric nonspecific porins of gram-negative bacteria. The capacity of outer membrane protein (OMP) preparations of P. multocida to bind to respiratory mucosal surface preparations was inhibited significantly by using a polyclonal anti-p35 antiserum in an adhesion ELISA. Anti-p35 antiserum cross-reacted with a 44 kDa (p44) MOMP of P. haemolytica. N-terminal sequencing of MOMP p44 revealed a homology of 81% with the putative porin MOMP p35 of P. multocida.

LC/MS and LC/MS/MS Screening for the Sites of Post-Translational Modification in Proteins

Coupled high performance liquid chromatography/mass spectrometry (LC/MS) is utilized for the molecular weight determination of peptides from enzymatic digests of proteins. Methods designed to identify the individual peptides that contain sites of phosphorylation are described and demonstrated for the Lys C digest of the nicotinic acetylcholine receptor (AChR). The technique employs both the use of LC/MS and coupled LC/tandem mass spectrometry (LC/MS/MS) to screen for the neutral loss of phosphate from the peptides. The coupling of HPLC and mass spectrometry is accomplished through the use of an atmospheric pressure ionization source (API) and IonSpra® LC/MS interface on a triple quadrupole mass spectrometer.

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences.

Abstract Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82 500 ± 60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat /Km . Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki <1μM) and a preference for a hydrophobic residue at the P1 site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10−5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2+-binding domain with complete helix-loop-helix structure (“EF hand”). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and n-terminal microsequencing of unknown proteins

The in vivo organ distribution of i.v. injected drug carriers is strongly influenced by the adsorption of plasma proteins after i.v. injection, e.g. uptake by the mononuclear phagocytic system (MPS). 2-D PAGE could be established to analyze plasma protein adsorption patterns on polysaccharide-stabilized aqueous iron oxide dispersions used as contrast agents in Magnetic Resonance Imaging (MRI). After incubation in human plasma, centrifugation, a washing procedure and a solubilization step were carried out to obtain the proteins adsorbed onto these ultrasmall particles (65 nm in diameter). Patterns of adsorbed proteins were analyzed in dependence on the washing medium used, i.e. highly purified water, phosphate buffered saline and Krebs buffer pH 7.4. Conductivity and composition of the washing medium influenced the adsorption of IgG onto the particles, but had little effect on the other proteins present. IgG was strongly reduced when using the relatively high conductive buffers. The more stabilizing polysaccharide was desorbed the larger was the total amount of adsorbed proteins. Appearance of two unknown chains of spots in the range of appr. 92 kDa, accounting for appr. 10% and 2% of the overall detected protein amount, was observed only when using Krebs buffer during the washing process. Performing N-terminal microsequencing one unknown chain of spots could be identified as a dimer of fibrinogen gamma chains.

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803

Abstract Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus.

In vitro binding of Campylobacter jejuni/coli outer membrane preparations to INT 407 cell membranes.

Outer membrane fractions (OMs) of nine Campylobacter (C.) jejuni and two C. coli strains belonging to different serovars, from human and various animal origins, were extracted by treatment with sodium N-lauryl sarcosinate. Using n-octyl-β-d-glucopyranoside a 42-kDa protein and a flagella-enriched fraction were obtained. The capacity of the crude bacterial OM preparations, the purified 42-kDa protein and the flagella to bind to membranes of the human embryonic intestinal cell line INT 407 was tested by an enzyme-linked immunosorbent assay. The crude OM and the 42-kDa-enriched fraction were found to bind very well to the cell membranes, whereas the flagella preparation showed only a weak binding. Using monoclonal antibodies (mAbs) with HS 2-lipopolysaccharide (LPS) specificity, binding of crude HS 2 strain OM preparations to cell membranes was detected in a significant range, whereas with flagellin-specific mAbs binding of OMs and flagella to cell membranes was only detected to a very low extent. Binding of OMs to cell membranes was inhibited by preincubation of OMs with serovar-specific mouse hyperimmune serum, whereas on preincubation with mAbs directed against LPS or flagella binding was practically not inhibited. OMs extracted after pretreatment of the bacteria with proteinase K showed an altered SDS-PAGE pattern especially for the 42-kDa protein subunit and and their capacity to bind to cell membranes was significantly reduced. The binding was also reduced by preincubation of the OMs with l-fucose or d-mannose.