Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu²⁺ in the BCA Kit’s reagent B (4% cupric sulfate) in a manner similar to that of the protein.     

Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml⁻¹) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml⁻¹) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.     

Anti-fibrillogenic and fibril destabilizing effects of metal ions on cystatin fibrils

Metals such as Cu²⁺, Fe³⁺, and Zn²⁺ are major contributors to the biology of a brain in stages of health, aging, and disease because of their unique effects on both protein structures (misfolding) and oxidative stress. The relationship between metal ions and neurodegenerative diseases is very complicated. Our study highlights how metal ions influence amyloid formation at low pH and on preformed amyloid fibrils. By using thioflavin T assay, ANS fluorescence, Congo red assay, circular dichroism, and microscopy to elucidate the effects of Cu²⁺, Fe³⁺, and Zn²⁺ on goat brain cystatin (GBC) aggregation at low pH. Results showed that Cu²⁺ and Fe³⁺ inhibit fibril formation of GBC by promoting amorphous aggregates. However, Zn²⁺ exclusively promotes fibril formation at low pH, leading to the formation of more ordered aggregates. Furthermore, the combined results of these complementary methods also suggested that Cu²⁺ and Fe³⁺ destabilize the β-sheet secondary structure of preformed amyloid fibrils of GBC.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

A micro-scale method for determining relative metal-binding affinities of proteins

This article describes a quick and easy method for determining relative binding affinities between proteins and metal ions. The method is based on separating unbound metal ions from metal ions bound to protein by ultrafiltration using microcentrifuge ultrafiltration units. Bovine serum albumin (BSA) was used as the test protein and the relative affinity towards divalent metal ions was found to be Cu²⁺>Zn²⁺>Cd²⁺>Pb²⁺>Ni²⁺>Co²⁺, which corresponds to the relative orders reported in the literature.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems

In the current study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and bicinchoninic acid (BCA) assays. Each phase-forming component above 1 and 5 wt% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing poly(ethylene glycol) (PEG)/salt concentration and PEG molecular weight. Therefore, a convenient dilution of both components (up to 1 and 5 wt%) before protein quantification is recommended in both assays, respectively, where the BCA assay is favored in comparison with the Bradford assay.     

Influence of divalent copper, manganese and zinc ions on fibril nucleation and elongation of the amyloid-like yeast prion determinant Sup35p-NM

There is a large body of evidence that divalent metal ions, particularly copper, might play a role in several protein folding pathologies like Alzheimer’s disease, Parkinson’s disease or the prion diseases. However, contribution of metal ions on pathogenesis and their molecular influence on the formation of amyloid structures is not clear. Therefore, the general influence of metals on the formation of amyloids is still controversially discussed. We have utilized the well established system of yeast Sup35p-NM to investigate the role of three different metal ions, Cu²⁺, Mn²⁺ and Zn²⁺, on amyloidogenesis. Recently, it has been shown that the prion determining region NM of the Saccharomyces cerevisiae prion protein Sup35p, which is responsible for the yeast prion phenotype [PSI⁺], specifically binds Cu²⁺ ions. We further characterized the affinity of NM for Cu²⁺, which were found to be comparable to that of other amyloidogenic proteins like the mammalian prion protein PrP. The specific binding sites could be located in the aminoterminal N-region which is known to initiate formation of amyloidogenic nuclei. In the presence of Cu²⁺, fibril nucleation was significantly delayed, probably due to influences of copper on the oligomeric ensemble of soluble Sup35p-NM, since Cu²⁺ altered the tertiary structure of soluble Sup35p-NM, while no influences on fibril elongation could be detected. The secondary structure of soluble or fibrous protein and the morphology of the fibrils were apparently not altered when assembled in presence of Cu²⁺. In contrast, Mn²⁺ and Zn²⁺ did not bind to Sup35p-NM and did not exhibit significant effects on the formation of NM amyloid fibrils.     

Copper uptake induces self-assembly of 18.5 kDa myelin basic protein (MBP)

Myelin basic protein (MBP) is predominantly found in the membranes of the myelin sheath of the central nervous system and is involved in important protein-protein and protein-lipid interactions in vivo and in vitro. Furthermore, divalent transition metal ions, especially Zn²⁺ and Cu²⁺, seem to directly affect the MBP-mediated formation and stabilization of the myelin sheath of the central nervous system. MBP belongs to the realm of intrinsically disordered proteins, and only fragmentary information is available regarding its partial structure(s) or supramolecular arrangements. Here, using standard continuous wave and modern pulse electron paramagnetic resonance methods, as well as dynamic light scattering, we demonstrate the uptake and specific coordination of two Cu²⁺ atoms or one Zn²⁺ atom per MBP molecule in solution. In the presence of phosphates, further addition of divalent metal ions above a characteristic threshold of four Cu²⁺ atoms or two Zn²⁺ atoms per MBP molecule leads to the formation of large MBP aggregates within the protein solution. In vivo, MBP-MBP interactions may thus be mediated by divalent cations.     

The Effect of Copper and Lead Ions on Growth and Lipid Composition of the Fern Matteuccia sthruthiopteris

In the present study, the effect of copper (Cu²⁺) and lead (Pb²⁺) ions on the growth and lipid composition of various parts of the fern, Matteuccia sthruthiopteris, was examined. Plants were incubated in the presence or absence of 1, 10, 100 μM of Cu(NO₃)₂ or Pb(NO₃)₂. Cu²⁺ and Pb²⁺ ions at concentrations of 1 and 10 μM caused an increased growth of the roots and leaves. A higher concentration of Pb²⁺ did not show any effect on growth, whereas that of Cu²⁺ slowed down the growth of the whole plants. The roots accumulated more than 700 μg of Cu²⁺ and 400 μg of Pb²⁺ per 1 g dry weight when the plants were incubated with the higher concentrations of metals, whereas in the leaves the concentration of Cu²⁺ was much lower and did not exceed 12 μg/g dry weight. No accumulation of Pb²⁺ ions by leaves was detected. The lipid composition of photosynthetic leave tissues was shown to be affected by the presence of metal ions in the root medium at either concentration studied. Various changes in lipid classes were noted as responsive reactions of M. sthruthiopteris to the heavy metal ions in nutrient medium. Cu²⁺ ions decreased the content of total lipids, total phospholipids, and individual phosphatidylcholines and phosphatidylethanolamines, whereas Pb²⁺ ions caused a decrease in the content of total lipids and glycolipids. Changes in the lipid composition were more pronounced in the mature leaves than in the scrolls of the studied fern.     

Contributions of cell wall and metal-binding peptide to Cd- and Cu-tolerances in suspension-cultured cells of tomato

Possible roles of cell wall and cytoplasmic peptides in the tolerance of cells to Cu²⁺ and Cd²⁺ ions were studied in suspension-cultured cells of tomato (Lycopersicon esculentum L. cv. Palace). Cu²⁺ and Cd²⁺ ions inhibited growth of wild type cells at concentrations more than 100 and 200 μM, respectively. Tomato cells readily developed tolerance to Cd²⁺ ions up to 1 mM but not to Cu²⁺ ions, after repeated subculturings in the presence of the respective ions. Such a metal-specific adaptation of cells was not due to the difference in the total uptakes between Cd²⁺ and Cu²⁺ ions by cells. Wild-type cells accumulated Cd²⁺ preferentially into the cytoplasmic peptide fraction and Cu²⁺ into the cell-wall fraction, when grown under the subtoxic metal conditions. Under excess metal conditions, Cd-tolerant cells produced greater amounts of Cd-binding peptides in the cytoplasm and retained lesser amounts of Cd²⁺ ions in the cell wall than did wild-type cells. In contrast, tomato cells grown in the presence of Cu²⁺ ions synthesized no detectable amounts of Cu-binding peptides in the cytoplasm and retained most of the Cu²⁺ in the cell-wall fraction, irrespective of cell lines. These results suggested that the cytoplasmic peptides rather than cell wall properties have a primary role in the response of tomato cells to excess metal environments.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of <Superscript>15</Superscript>N longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR

Magic-angle spinning solid-state NMR measurements of ¹⁵N longitudinal paramagnetic relaxation enhancements (PREs) in ¹³C,¹⁵N-labeled proteins modified with Cu²⁺-chelating tags can yield multiple long-range electron-nucleus distance restraints up to ~20 Å (Nadaud et al. in J Am Chem Soc 131:8108–8120, 2009). Using the EDTA-Cu²⁺ K28C mutant of B1 immunoglobulin binding domain of protein G (GB1) as a model, we investigate the effects on such measurements of intermolecular electron-nucleus couplings and intrinsic metal binding sites, both of which may potentially complicate the interpretation of PRE data in terms of the intramolecular protein fold. To quantitatively assess the influence of intermolecular ¹⁵N-Cu²⁺ interactions we have determined a nearly complete set of longitudinal ¹⁵N PREs for a series of microcrystalline samples containing ~10, 15 and 25 mol percent of the ¹³C,¹⁵N-labeled EDTA-Cu²⁺-tagged protein diluted in a matrix of diamagnetic natural abundance GB1. The residual intermolecular interactions were found to be minor on the whole and account for only a fraction of the relatively small but systematic deviations observed between the experimental ¹⁵N PREs and corresponding values calculated using protein structural models for residues furthest removed from the EDTA-Cu²⁺ tag. This suggests that these deviations are also caused in part by other factors not related to the protein structure, such as the presence in the protein of intrinsic secondary sites capable of binding Cu²⁺ ions. To probe this issue we performed a Cu²⁺ titration study for K28C-EDTA GB1 monitored by 2D ¹⁵N-¹H solution-state NMR, which revealed that while for Cu²⁺:protein molar ratios of ≤ 1.0 Cu²⁺ binds primarily to the high-affinity EDTA tag, as anticipated, at even slightly super-stoichiometric ratios the Cu²⁺ ions can also associate with side-chains of aspartate and glutamate residues. This in turn is expected to lead to enhanced PREs for residues located in the vicinity of the secondary Cu²⁺ binding sites, and indeed many of these residues were ones found to display the elevated longitudinal ¹⁵N PREs in the solid phase.     

Mechanisms underlying iron and copper ions toxicity in biological systems: Pro-oxidant activity and protein-binding effects

Iron and copper ions, in their unbound form, may lead to the generation of reactive oxygen species via Haber–Weiss and/or Fenton reactions. In addition, it has been shown that copper ions can irreversibly and non-specifically bind to thiol groups in proteins. This non-specific binding property has not been fully addressed for iron ions. Thus, the present study compares both the pro-oxidant and the non-specific binding properties of Fe³⁺ and Cu²⁺, using rat liver cytosol and microsomes as biological systems. Our data show that, in the absence of proteins, Cu²⁺/ascorbate elicited more oxygen consumption than Fe³⁺/ascorbate under identical conditions. Presence of cytosolic and microsomal protein, however, differentially altered oxygen consumption patterns. In addition, Cu²⁺/ascorbate increased microsomal lipid peroxidation and decreased cytosolic and microsomal content of thiol groups more efficiently than Fe³⁺/ascorbate. Finally, Fe³⁺/ascorbate and Cu²⁺/ascorbate inhibited in different ways cytosolic and microsomal glutathione S-transferase (GST) activities, which are differentially sensitive to oxidants. Moreover, in the absence of ascorbate, only Cu²⁺ decreased the content of cytosolic and microsomal thiol groups and inhibited cytosolic and microsomal GST activities. Catechin partially prevented the damage to thiol groups elicited by Fe³⁺/ascorbate and Cu²⁺/ascorbate but not by Cu²⁺ alone. N-Acetylcysteine completely prevented the damage elicited by Cu²⁺/ascorbate, Fe³⁺/ascorbate and Cu²⁺ alone. N-Acetylcysteine also completely reversed the damage to thiol groups elicited by Fe³⁺/ascorbate, partially reversed that of Cu²⁺/ascorbate but failed to reverse the damage promoted by Cu²⁺ alone. Our data are discussed in terms to the potential damage that the accumulation of iron and copper ions can promote in biological systems.     

Copper Ion from Cu2O Crystal Induces AMPK-Mediated Autophagy via Superoxide in Endothelial Cells

Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions (Cu⁺ and Cu²⁺), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped Cu₂O and CuO crystals were prepared to test the role of the two different ions, Cu⁺ and Cu²⁺, respectively. The Cu₂O crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The Cu₂O crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. Cu₂O crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit Cu₂O-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of Cu⁺ ions in the vascular system, where Cu⁺ induces autophagy while Cu²⁺ has no detected effect.     

Copper Reduction and Contact Killing of Bacteria by Iron Surfaces

The well-established killing of bacteria by copper surfaces, also called contact killing, is currently believed to be a combined effect of bacterial contact with the copper surface and the dissolution of copper, resulting in lethal bacterial damage. Iron can similarly be released in ionic form from iron surfaces and would thus be expected to also exhibit contact killing, although essentially no contact killing is observed by iron surfaces. However, we show here that the exposure of bacteria to iron surfaces in the presence of copper ions results in efficient contact killing. The process involves reduction of Cu²⁺ to Cu⁺ by iron; Cu⁺ has been shown to be considerably more toxic to cells than Cu²⁺. The specific Cu⁺ chelator, bicinchoninic acid, suppresses contact killing by chelating the Cu⁺ ions. These findings underline the importance of Cu⁺ ions in the contact killing process and infer that iron-based alloys containing copper could provide novel antimicrobial materials.     

Binding of water to "types I and II" Cu2+ in proteins

Water proton spin-lattice relaxation times have been measured at 30MHz between 280 – 333 K in aqueous solutions of proteins containing Type I Cu²⁺ ions (azurin and umecyanin) and Type II Cu²⁺ ions (benzylamine oxidase and superoxide dismutase). These measurements show that Type II Cu²⁺ is accessible to exchangeable water molecules but Type I is not. This behaviour is consistent with the EPR and optical properties of these ions and their likely biochemical functions.