Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

Structure of alginate gels: interaction of diuronate units with divalent cations from density functional calculations

The complexation of (1→4) linked α-L-guluronate (G) and β-D-mannuronate (M) disaccharides with Mg(2+), Ca(2+), Sr(2+), Mn(2+), Co(2+), Cu(2+), and Zn(2+) cations have been studied with quantum chemical density functional theory (DFT)-based method. A large number of possible cation-diuronate complexes, with one and two GG or MM disaccharide units and with or without water molecules in the inner coordination shells have been considered. The computed bond distances, cation interaction energies, and molecular orbital composition analysis revealed that the complexation of the transition metal (TM) ions to the disaccharides occurs via the formation of strong coordination-covalent bonds. On the contrary, the alkaline earth cations form ionic bonds with the uronates. The unidentate binding is found to be the most favored one in the TM hydrated and water-free complexes. By removing water molecules, the bidentate chelating binding also occurs, although it is found to be energetically less favored by 1 to 1.5 eV than the unidentate one. A good correlation is obtained between the alginate affinity trend toward TM cations and the interaction energies of the TM cations in all studied complexes, which suggests that the alginate affinities are strongly related to the chemical interaction strength of TM cations-uronate complexes. The trend of the interaction energies of the alkaline earth cations in the ionic complexes is opposite to the alginate affinity order. The binding strength is thus not a limiting factor in the alginate gelation in the presence of alkaline earth cations at variance with the TM cations.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.     

Detection of heavy metal ions using protein-functionalized microcantilever sensors

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg(2+) and Zn(2+). AgNt84-6, a protein that has the ability to bind multiple atoms of Ni(2+), Zn(2+), Co(2+), Cu(2+), Cd(2+) and Hg(2+) was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl(2) and 0.1 mM ZnCl(2) solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl(2) solution did not result in a similar bending indicating a weak or no interaction of Mn(2+) ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn(2+) ions but not with Mn(2+) ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.     

Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Influence of oligoguluronates on alginate gelation, kinetics, and polymer organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.     

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Sol–gel transition of alginate solution by the addition of various divalent cations: 13C-nmr spectroscopic study

¹³C-NMR spectroscopic studies have been made on alginate solutions undergoing sol–gel transition induced by four different divalent cations: Ca, Cu, Co, and Mn. From the analysis of nmr spectra and relaxation times, we have found different interaction modes existing between the Ca–alginate systems and the transition metal (Cu, Co, and Mn)–alginate systems. In the Ca–alginate systems, there exists a specific interaction characterized by a strong autocooperative binding between guluronate residues and calcium ions, and all functional groups in guluronate residues are considered to involve the interaction with calcium ions. On the other hand, in transition metal (Cu, Co, and Mn)–alginate systems, sol–gel transition is characterized by a complex formation in which the carboxyl groups in both mannuronate and guluronate residues are coordinated to metal ions. The other functional groups, like hydroxyl groups, do not participate in the binding to metal ions. It is suggested by relaxation time measurements that from a microscopic point of view the sol–gel transition phenomena can be explained as a dynamic process in which the low frequency molecular motions are dominant and increase their proportions with the formation of three-dimensional cross-links. © 1993 John Wiley & Sons, Inc.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels

Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.     

Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads

Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.     

Picogram detection of metal ions by melanin-sensitized piezoelectric sensor

A heavy metal ion sensor was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM). A mercury ion sensitivity of 518+/-37 Hz/ppm was observed, a substantial increase in sensitivity compared to previous reports of 10-50 Hz/ppm with the limit of detection at 5 ppb. Detection of other metal ions including Sn(2+), Ge(4+), Li(+), Zn(2+), Cu(2+), Bi(3+), Co(2+), Al(3+), Ni(2+), Ag(+), and Fe(3+) were also performed. Unexpectedly, binding of Mn(7+), Pb(2+), Cd(2+), and Cr(3+) increased resonant frequencies. The surface profile of melanin thin film upon binding to metal ions was investigated by atomic force microscopy (AFM). Structural change of melanin upon binding to metal ions was characterized by circular dichroism and by infrared spectroscopy. The current study provides the first example of melanin-coated piezoelectric sensor showing high sensitivity and selectivity to metal ions.     

Effect of metal ions on de novo aggregation of full-length prion protein

It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu(2+), Mn(2+), Ni(2+), Co(2+), and Zn(2+)) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn(2+) at low micromolar concentrations that could be blocked by nanomolar concentration of Cu(2+). These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.