The pH dependent properties of metallotransferrins: a comparative study

The dependence on pH of the absorption and circular dichroic spectra of iron(III), cobalt(III) and copper(II) transferrins has been (re)investigated. In the alkaline region, the CD profiles of iron(III) and cobalt(III) transferrin are essentially pH independent up to pH 11; only for very high pH values (pH > 11) is breakdown of the cobalt(III) and iron(III) transferrin derivatives observed, without evidence of conformational rearrangements. By contrast, the CD profiles of copper transferrin show drastic changes in shape around pH 10; these spectral changes, which are fitted to a pK_a of ~10.4, are interpreted in terms of a substantial rearrangement of the local environment of the copper ions at high pH. Although the CD spectra of copper transferrin at alkaline pH strictly resemble those observed upon addition of modifier anions, the mechanism of site destabilization in the two cases is different; at variance with the case of modifier anions, our results suggest that the high pH form of copper transferrin still contains the synergistic anion. A¹³C NMR experiment has confirmed this view. In the acidic region, iron(III) and cobalt(III) transferrins are stable down to pH ~6. For lower pH values progressive metal detachment is observed without evidence of conformational changes; around pH 4.5 most bound metals are released. In the case of the less stable copper-transferrin, metal removal from the specific binding sites is already complete around pH 6.0; in concomitance with release from the primary sites, binding of copper ions to secondary sites is observed. Additional information has been gained from CD experiments in the far UV. The pH dependent properties of iron(III), cobalt(III) and copper(II) transferrin are discussed in the frame of the present knowledge of transferrin chemistry, particular emphasis being attributed to the comparison between tripositive and bipositive metal derivatives.     

Non-cooperative Ca(II) removal and terbium(III) substitution in carp muscle calcium binding parvalbumin.

Close coorelation of atomic absorption measurements for Ca(II) contents indicates that from pH 5.8-7.4 a twentyfold excess of EGTA1 removes but one of two Ca(II) from carp parvalbumin. Thus binding of the two Ca(II) appears to be noncooperative. The maximum in emission intensity observed at a nonintegral 1.4-1.7 equivs of added Tb(III) is shown to be due to quenching by excess Tb(III). The emission intensity at the maximum increased 40% upon dialysis to remove Tb(III) not bound in the CD or EF sites. Atomic absorption results show that both Ca(CD) and Ca(EF) of native parvalbumin are easily replaced by Tb(III). Emission of Tb(EF) is not quenched by Tb(CD), but by solution Tb(III) bound at a third site, perhaps the single water molecule bound to Tb(EF). Labeling of the single sulfhydryl group with a trifluoroacetonyl gorup yields a protein with ultraviolet circular dichroism, emission, and circularly polarized emission spectra closely similar to those of native parvalbumin.     

Paramecium secretory granule content: quantitative studies on in vitro expansion and its regulation by calcium and pH

Ca2+-dependent secretion in Paramecium involves the exocytic release of a paracrystalline secretory product, the trichocyst matrix, which undergoes a characteristic structural change from a highly condensed storage form (Stage I) to an extended needle-like structure (Stage III) during release. We studied trichocyst matrix expansion in vitro to examine factors regulating the state of secretory organelle content. A new method for the isolation of membrane-free, condensed (Stage I) trichocyst matrices is described. These highly purified, condensed matrices were used to develop a rapid quantitative, spectrophotometric assay for matrix expansion to examine factors regulating the Stage I and Stage III transition. Expansion from Stages I to III was elicited in vitro by addition of Ca2+ and we found that at neutral pH, expansion required a Ca2+ concentration slightly above 10(-6)M. Previous studies indicate that calmodulin (CaM) antagonists inhibit matrix expansion in vivo. However, in vitro matrix expansion is normal even when trichocyst matrices are preincubated in CaM antagonists before stimulation. Thus, matrix components themselves are unlikely to be the site of CaM antagonist action in vivo. In vitro matrix expansion is also modulated by pH. Decreasing pH to 6.0 inhibits expansion, i.e., expansion requires higher Ca2+ concentration. Conversely, increasing pH to greater than 7.0 promotes expansion, allowing it to occur at a lower Ca2+ concentration. The pH sensitivity of the Ca2+ binding sites of the matrix suggests that, in vivo, the interior of the trichocyst vesicle may be maintained at an acidic pH. Exposure of cells to acridine orange, a fluorescent amine that accumulates in acidic intracellular compartments, leads to its uptake and concentration within trichocysts. Thus intratrichocyst pH appears to be acidic in vivo and may serve as a regulatory or "safety" mechanism to inhibit premature expansion.     

Structural polymorphism of telomeric DNA regulated by pH and divalent cation

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.     

Chemical modifications of histidyl and tyrosyl residues of inorganic pyrophosphatase from Escherichia coli

Chemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3.6.1.1] from Escherichia coli Q13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.     

Circular dichroism studies on coat proteins of some strains and mutants of tobacco mosaic virus

1. Tobacco mosaic virus (TMV) protein has in near ultraviolet a complex but well resolved circular dichroism (CD) spectrum at room temperature. There are seven positive bands at 248, 252, 257, 265, 274, 281 and 291 nm, and a negative one at 296 nm. The CD spectrum is pH-dependent. The shape of the pH-dependence curves and the comparison with CD spectra of model compounds suggest that the bands at 248, 252 and 257 nm are mainly caused by phenylalanyl, those at 265, 274 and 281 nm by tyrosyl, and those at 291 and 296 nm by tryptophanyl side chains. 2. Only insignificant changes of the tertiary structure seem to occur between pH 6.5 and 8.5. Changes in ellipticity of TMV protein during the pH-induced polymerization reaction suggest that: (1) tyrosyl residues are involved in the binding of subunits, (2) phenylalanyl residues seem to be transferred to a less rigid environment, and (3) tryptophanyl residues are not essential for the reaction. 3. The proteins of several TMV strains and mutants studied have similar far ultraviolet CD spectra and apparently do not differ significantly in their structure. Their near ultraviolet CD spectra are, however, different. Replacements involving aliphatic amino acids do not change considerably the near ultraviolet CD spectra. On the other hand, replacements involving aromatic amino acids have a great effect on the spectra rendering possible identification of CD bands and recognition of the aromatic amino acid residues responsible for optical activity.     

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.     

Interaction of parvalbumin of pike II with calcium and terbium ions

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from F?rster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.     

Study of Ca2+-binding centers of parvalbumin by the distant fine structure of x-ray absorption spectra

An investigation of Ca2+-binding centers of parvalbumin II and III by analysing distant fine structure of X-ray absorption spectra of metal was performed. Protein preparations of parvalbumin II and III in which Ca2+ was isomorphically replaced by Tb3+ were studied. For spectra analyses a standard method of Fourier transformation was used. The middle of the first absorption maximum was taken as origin for energy calculations. Comparison of spectra and modules of Fourier transformations for normalized oscillations of the X-ray spectra of absorption of the II and III components, revealed that the spectra and Fourier-transformants coincide in the 2--6 A interval. This allows to infer the coincidence of the coordinate numbers, average interatomic distances and their dispersions in Ca2+-binding centers of the two protein components.     

Vacuum ultraviolet circular dichroism as an indicator of helical handedness in nucleic acids.

Calculated circular dichroism spectra are presented for double-stranded polynucleotides of regular sequences in A-RNA, A-DNA, B-DNA, and Z-DNA conformations. Quantum mechanical matrix method calculations were carried out in the near and vacuum ultraviolet regions. In the near UV, the calculated spectra agreed qualitatively with the measured spectra. However in the far and vacuum UV, the calculated CD compared nearly quantitatively with the experimental spectra. The calculations show that the sign of the CD in the vacuum UV, in contrast to that in the near UV, can be correlated with the handedness of the helix.     

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.     

Triggering of Ca2+ signals by NAADP-gated two-pore channels: a role for membrane contact sites?

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger implicated in many Ca2+-dependent cellular processes. It is highly unusual in that it appears to trigger Ca2+ release from acidic organelles such as lysosomes. These signals are often amplified by archetypal Ca2+ channels located in the endoplasmic reticulum. Recent studies have converged on the TPCs (two-pore channels) which localize to the endolysosomal system as the likely primary targets through which NAADP mediates its effects. 'Chatter' between TPCs and endoplasmic reticulum Ca2+ channels is disrupted when TPCs are directed away from the endolysosomal system. This suggests that intracellular Ca2+ release channels may be closely apposed, possibly at specific membrane contact sites between acidic organelles and the endoplasmic reticulum.     

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.     

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.     

Global structural changes in annexin 12. The roles of phospholipid,Ca2+, and pH

Ca2+-dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo Ca2+-independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca2+-dependent membrane-bound form at neutral pH. In contrast, all Ca2+-independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca2+-dependent membrane form were >25 A apart in the Ca2+-independent membrane-bound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca2+-independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca2+-independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca2+-independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca2+-independent membrane interaction in vivo.     

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.     

The phenoloxidases of the ascomycete Podospora anserina. XI. The state of copper of laccases I, II and III.

1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.     

Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three Ca2+-binding proteins from porcine liver and intestine differ immunologically and physicochemically and are distinct in Ca2+ affinities

Intestinal brush-border-derived membrane vesicles contain, after demembranation in the presence of Ca2+, a subset of polypeptides that are specifically solubilized by the addition of Ca2+ chelators. As described previously, this fractionation scheme leads to the enrichment of two major proteins (I and II), one of which has been shown to be identical to the cellular p36K target of Rous sarcoma virus-encoded tyrosine-specific protein kinase (Gerke, V., and Weber, K., (1984) EMBO J. 3, 227-233). We have applied a similar protocol to membrane vesicles from porcine liver and purified a third Ca2+-binding protein (III). All three proteins had wide tissue distributions, and were absent from brain, red blood cells, and cardiac and skeletal muscle. Relative amounts varied between tissues, with protein I low in liver and protein III very low in intestine. Despite their similar extractability the three proteins (I, II, and III) are clearly distinct as far as immunological, biochemical, and physicochemical properties are concerned. They also show characteristic differences in their affinities for Ca2+ ions. The association constants of Ca2+ binding for proteins I and III have been estimated by means of indirect methods to be 10(4) M-1 (protein I) and 10(6) M-1 (protein III), while the direct Hummel-Dreyer method reveals Ca2+ binding to protein II, characterized by an association constant of 0.4 X 10(5) M-1 in the absence and 0.2 X 10(5) M-1 in the presence of 2 mM MgCl2. Conformational changes upon binding Ca2+ are described for protein II using circular dichroism, fluorescence emission, and UV difference spectra. These alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, and led to increased hydrophobicity of protein II that would explain the observed Ca2+-dependent interaction with hydrophobic matrices like phenyl-Sepharose.