The effect of copper and zinc at neutral and acidic pH on the general haematology and osmoregulation of Oreochromis mossambicus

The objective of this study was to determine the effect of sublethal copper and zinc concentrations at a neutral and an acidic pH, on selected haematological parameters as well as on the total osmolality and electrolyte concentrations of Oreochromis mossambicus. In general, at neutral pH copper and zinc caused blood acidosis, increases in circulating white blood cell numbers, causing stimulation of the immune system, and a rapid release of red blood cells from haemopoietic tissue, as reflected in the decreases in mean corpuscular volume. Increases in red blood cells are attributed to an increase in the oxygen-carrying haemoglobin as an adaptation to altered respiratory homeostasis caused by copper and zinc. These increases are therefore a secondary reaction to the metals and not the result of direct stimulation of the haemopoietic tissue. In contrast, at an acidic pH copper and zinc concentrations usually caused blood alkalosis and decreases in white blood cell numbers, due to the bioconcentration of metals which blocks and suppresses the leucopoietic tissue. Decreases were also recorded in red blood cell counts, haemoglobin, haematocrit and mean corpuscular volume, which can be ascribed to anaemic and hypoxic conditions, gill damage and impaired osmoregulation.     

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.     

Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions.

The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2+ concentration are considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycine-conjugated bile acids because of metastability, whereas super-saturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing Ca2+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD

Copper(II) and zinc(II) complexes of the peptides Ac-HisValHis-NH2 and Ac-HisValGlyAsp-NH2 related to the active site of the enzyme CuZnSOD were studied by potentiometric and spectroscopic (UV-Vis, CD and EPR) techniques. The results reveal that both ligands have effective metal binding sites, but the tripeptide is a much stronger complexing agent than the tetrapeptide. The formation of a macrochelate via the coordination of the imidazolyl residues is suggested in the copper(II)-Ac-HisValHis-NH2 system in the acidic pH range, while a 4N complex predominates at physiological pH. The interaction of Ac-HisValHis-NH2 with zinc(II) results in the formation of a precipitate indicating polynuclear complex formation. Both copper(II)-Ac-HisValHis-NH2 and copper(II)-HisValHis systems exhibit catalytic activity toward the dismutation of superoxide anion at physiological pH, but the saturated coordination sphere of the metal ions in both systems results in low reactivity as compared to the native enzyme.     

Effects of controlled pH on organic and inorganic composition in haemolymph,epidermal tissue and cuticle of mud crab Scylla serrata

Analysis of organic and inorganic compounds in plasma, epidermal tissue and cuticle were accomplished in the intermolt (C3 stage) of crab Scylla serrata incubated in different pH media. Significant changes with similar trends for protein, carbohydrates, glycosaminoglycans (GAG), sulphur, calcium, magnesium, potassium, phosphorus and copper in the plasma suggested higher dissolution in an acidic medium while the deposition increased in alkaline medium. Similar decreases in protein, carbohydrate and GAG in the epidermal compartment were observed from pH 4 to pH 12. However, significantly higher contents of sodium, chloride, potassium, phosphorus, magnesium, sulphur and copper were measured at pH 7.5 with a symmetrical decrease profile in both acidic and alkaline media, resulting from synergistic effects in the osmotic regulation. Clear changes in calcium concentrations were observed with a sharp increase from lower contents at pH 7.5 to higher at pH 12. In the cuticle, the acidic condition induced a significant dissolution of HCl-protein, GAG, calcium and magnesium contents. On the other hand, the alkaline condition induced a significant decrease in carbohydrate, calcium, chloride, sulphur and potassium. A reduction trend is seen for NaOH and H(2)O-protein contents in the cuticle. These observations suggest that GAG and HCl-protein might constitute the most soluble fraction with high affinity for calcium binding and easily removed in acidic conditions. Additionally, it is possible to speculate that the carbohydrates associated with the NaOH and H(2)O-proteins may form an interface between the soluble matrix fraction and the chitin framework. Sulphur groups seem to present a strong linkage role in this interface fraction, maybe only broken by a specific enzyme in extreme alkaline conditions with subsequent release of significant calcium from the shell.     

Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternae of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd approximately 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.     

A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

Effect of pH on the speciation and solubility of divalent metals in human and bovine milks

Computer models estimated the ligand speciation and solubility of calcium, magnesium, zinc, and copper over a pH range for low molecular weight fractions characteristic of either human or bovine milks. Above pH 4 calcium is the only metal predicted to precipitate. Most of the remaining soluble calcium, magnesium, and zinc should be complexed with citrate. The solubility of calcium, magnesium, and zinc in human and bovine milks was measured experimentally from pH 2 to 7. The solubility of all three metals decreased as the pH increased. Calcium and zinc were soluble over a narrower pH range in bovine milk than in human milk. Increasing the levels of either calcium or inorganic phosphate alone in decaseinated human milk did not affect the solubility of zinc, but when both calcium and inorganic phosphate were added at levels comparable to bovine milk the solubility of zinc decreased at the higher pH's. The decreased solubility of zinc in skimmed milks in pH's characteristic of the small intestine is likely due to coprecipitation of zinc with calcium phosphate--a reaction not predicted for milk systems from known chemical solubility product data.     

The effect of pH and nitrite on the haem pocket of GLB-33, a globin-coupled neuronal transmembrane receptor of Caenorhabditis elegans

Out of the 34 globins in Caenorhabditis elegans, GLB-33 is a putative globin-coupled transmembrane receptor with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of electronic circular dichroism, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.     

Annexin A4 binding to anionic phospholipid vesicles modulated by pH and calcium

Annexin A4 belongs to a class of Ca(2+)-binding proteins for which different functions in the cell have proposed, e.g. involvement in exocytosis and in the coagulation process. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study the interaction of annexin A4 with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) or from phosphatidic acid (PA) is investigated at neutral and acidic pH. Annexin A4 strongly binds to either lipid at acidic pH, whereas at neutral pH only weak binding to PA and no binding to PS occurs. Addition of 40 microM Ca(2+) leads to a strong binding to the lipids also at neutral pH. This is caused by the different electric charge of the protein below and above its isoelectric point. Binding of annexin A4 induces dehydration of the vesicle surface. The strength of the effects is much greater at pH 4 than at pH 7.4. At pH 7.4 annexin A4 reduces the Ca(2+)-threshold concentration necessary to induce fusion of PA LUV. The Ca(2+) induced fusion of PS LUV is not affected by annexin A4 at pH 7.4. At pH 4 annexin A4 induces fusion of either vesicles without Ca(2+). Despite the low binding extents at neutral pH annexin A4 induces a Ca(2+) independent leakage of PS- or PA-LUV. The leakage extent is increased at acidic pH. From the data two suggestions are made: (1) At pH 4 annexin A4 (at least partially) penetrates into the bilayer in contrast to the preferred location at the vesicle surface at neutral pH. The conformation of annexin A4 seems to be different at the two conditions. (2) At neutral pH, Annexin A4 seems to be able to bind two PA vesicles simultaneously; however, only one PS vesicle at the same time. This behavior might be related to a recently described double Ca(2+) binding site, which appears to be uniquely suited for PS.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH

The novel tetrameric structure of human beta-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human beta-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-beta-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to beta-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-beta-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 beta-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts beta-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.     

Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase.

The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton.     

Crystal structures of the peanut lectin-lactose complex at acidic pH: retention of unusual quaternary structure, empty and carbohydrate bound combining sites, molecular mimicry and crystal packing directed by interactions at the combining site

The crystal structures of a monoclinic and a triclinic form of the peanut lectin-lactose complex, grown at pH 4.6, have been determined. They contain two and one crystallographically independent tetramers, respectively. The unusual "open" quaternary structure of the lectin, observed in the orthorhombic complex grown in neutral pH, is retained at the acidic pH. The sugar molecule is bound to three of the eight subunits in the monoclinic crystals, whereas the combining sites in four are empty. The lectin-sugar interactions are almost the same at neutral and acidic pH. A comparison of the sugar-bound and free subunits indicates that the geometry of the combining site is relatively unaffected by ligand binding. The combining site of the eighth subunit in the monoclinic crystals is bound to a peptide stretch in a loop from a neighboring molecule. The same interaction exists in two subunits of the triclinic crystals, whereas density corresponding to sugar exists in the combining sites of the other two subunits. Solution studies show that oligopeptides with sequences corresponding to that in the loop bind to the lectin at acidic pH, but only with reduced affinity at neutral pH. The reverse is the case with the binding of lactose to the lectin. A comparison of the neutral and acidic pH crystal structures indicates that the molecular packing in the latter is directed to a substantial extent by the increased affinity of the peptide loop to the combining site at acidic pH.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Effect of pH on tolerance of Hormidium rivulare to zinc and copper

Summary The toxicity of zinc to a population of Hormidium rivulare isolated from an acid mine drainage was shown to be least at the optimum pH range for the growth of the alga, pH 3.5–4.0; toxicity increases markedly at higher pH values. Calcium clearly antagonizes the toxicity of zinc. Populations of H. rivulare isolated from higher pH values and which are resistant to zinc, are also especially resistant to low pH values, although they are unlikely ever to encounter such values naturally. Nevertheless raised levels of calcium bring about only a slight improvement of growth at very low pH values in the absence of zinc, so the mechanisms of pH and zinc tolerance are not the same. Although the acid stream population grows in the field in an environment with rather similar levels of zinc and copper, copper is less toxic than zinc at pH 3.5, but much more toxic than zinc at pH 6.0.     

Influence of pH on the activity of thrombin-derived antimicrobial peptides

The wound environment is characterized by physiological pH changes. Proteolysis of thrombin by wound-derived proteases, such as neutrophil elastase, generates antimicrobial thrombin-derived C-terminal peptides (TCPs), such as HVF18 (HVFRLKKWIQKVIDQFGE). Presence of such TCPs in human wound fluids in vivo, as well as the occurrence of an evolutionarily conserved His residue in the primary amino acid sequence of TCPs, prompted us to investigate the pH-dependent antibacterial action of HVF18, as well as of the prototypic GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE). We show that protonation of this His residue at pH 5.5 increases the antibacterial activity of both TCPs against Gram-negative Escherichia coli by membrane disruption. Physiological salt level (150 mM NaCl) augments antibacterial activity of GKY25 but diminishes for the shorter HVF18. Replacing His with Leu or Ser in GKY25 abolishes the His protonation-dependent increase in antibacterial activity at pH 5.5, whereas substitution with Lys maintains activity at neutral (pH 7.4) and acidic pH. Interestingly, both TCPs display decreased binding affinities to human CD14 with decreasing pH, suggesting a likely switch in mode-of-action, from anti-inflammatory at neutral pH to antibacterial at acidic pH. Together, the results demonstrate that apart from structural prerequisites such as peptide length, charge, and hydrophobicity, the evolutionarily conserved His residue of TCPs influences their antibacterial effects and reveals a previously unknown aspect of TCPs biological action.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.