Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron-ion-containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate-covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)(4) (C/H/Y)(X)(4) to characterize peptide ligands with considerable hemin binding capacities. Some of the library-selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His-based (≈39 %) and Tyr-based (≈40 %) sequences over Cys-based ones (≈21 %) was detected. The binding affinities for the library-selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme-binding domain.

Spherical nanoparticles with sizes from 80 to 200 nm are obtained by self-assembly of highly functionalized 6-deoxy-6-(ω-aminoalkyl)aminocellulosecarbamates. The particles are very stable, nontoxic, and possess primary amino groups that are accessible to further modifications in aqueous suspension. The particles can be labeled with rhodamine B isothiocyanate without changing their size, stability, and shape. The nanoparticles obtained are investigated by means of photo correlation spectroscopy, zeta potential measurements, SEM and fluorescence spectroscopy. Incorporation of the nanoparticles in human foreskin fibroblasts BJ-1-htert and breast carcinoma MCF-7 cells without any transfection reagent is proved by means of confocal laser scanning microscopy.

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.

Many proteins form noncovalent and thermodynamically reversible oligomers, and the state of self-association can dictate a proteins functionality. DNA-binding proteins are very often dimeric, while other proteins exist as trimers (e.g. chloramphenicol transacetylase), tetramers (e.g. hemoglobin), or higher-order reversible association products (tubulin, viral coat proteins, sickle cell hemoglobin), with clear functional roles that have never been observed for carbohydrates. Although weak self-association in a polysaccharide has been shown, [1] we show for the first time the presence of multiple oligomeric forms in a whole class of polymeric carbohydrates, 6-deoxy-6-aminocelluloses, using the hydrodynamic technique of analytical ultracentrifugation as a probe.Water-soluble aminocelluloses were prepared by the reaction of tosyl cellulose with an excess of di-or trifunctional amines, namely with tris(2-aminoethyl)amine yielding 6-deoxy-6-(2-(bis(2-aminoethyl)aminoethylamino) (BAEA) cellulose (1-3), as depicted in Figure 1. Similar structures were obtained from reactions of tosyl cellulose with 1,2-diaminoethane and 4,7,10-trioxa-1,13-tridecandiamine to give 6-deoxy-6-(2-aminoethyl)amino (AEA) cellulose 4 and 6-deoxy-6-(13-amino-4,7,10-trioxatridecaneamino) (ATOTA) cellulose 5, respectively (Supporting Information Figure 1). The degree of substitution (DS) ranged from 0.60 to 0.85 (Supporting Information Table 1). NMR spectroscopic studies revealed that the nucleophilic displacement takes place at the primary position 6 of the anhydroglucose unit (AGU). [2] Sedimentation coefficient distributions for the five different 6-deoxy-6-aminocelluloses were obtained from sedimentation velocity experiments in the analytical ultracentrifuge for six different solute loading concentrations (from 0.125 to 2.0 mg mL À1 ) in 0.1m phosphate-buffered saline (pH 6.8). Astonishingly, for every aminocellulose studied, the sedimentation coefficient distributions show between four and five discrete species with a stepwise increase in sedimentation coefficient. For example, the lowest sedimentation coefficient of the BAEA cellulose 1, prepared from cellulose with a degree of polymerization (DP) of 450, was detected at 1.8 Svedbergs (S). Additional species sedimenting at peak maxima of approximately 2.8, 4.0, 5.1, and 6.5 S were also clearly found using the SEDFIT algorithm of Dam and Schuck [3] (Figure 1). The measurements were repeated for two other BAEA celluloses prepared from cellulose with DP = 250, and comparable results were obtained (Figure 2 b, c). Peaks with sedimentation coefficients of approximately 1.6, 2.3, 3.1, 4.3, and 5.3 S have been found for an AEA cellulose (4) prepared from cellulose with DP = 450. Furthermore, the ATOTA cellulose 5 shows peaks sedimenting with maxima at approximately 1.7, 2.9, 4.3, and 5.5 S (Figure 2; Supporting Information Table 2). To check that the peaks were not artifacts of the analysis procedure, least-squares g(s) versus s distribution data were also obtained (Supporting Information Figure 3 a...

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