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• W2004014208 abstract "Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 ± 0.01 nm, maximum diameter = 10.59 ± 0.01 nm, and volume = 255 ± 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1°C and 4 at 25°C, providing ΔH = −20 kcal/mol and ΔS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]−8. Equilibrium oxygen binding measurements at pH 6.5-8.5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9.5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25°C. P50 was decreased 3- and 2-fold in ~100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity. Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 ± 0.01 nm, maximum diameter = 10.59 ± 0.01 nm, and volume = 255 ± 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1°C and 4 at 25°C, providing ΔH = −20 kcal/mol and ΔS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]−8. Equilibrium oxygen binding measurements at pH 6.5-8.5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9.5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25°C. P50 was decreased 3- and 2-fold in ~100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity. The HBL 1The abbreviations used are: HBLhexagonal bilayerHbhemoglobinGdmClguanidinium chlorideKSiWpotassium undecatungstosilicateK8[SiW11O39].14H2O; NaAsWsodium tetra-contatungstotetraarsenate(III)Na26[BaAs4W40O140]·60H2O; BisTris2-bis(2-hydroxyethyl)-amino-2-(hydroxymethyl)-1,3-propanediolFPLCfast protein liquid chromatographyAUabsorbance unitr.m.s.root mean squareEMGexponentially modified gaussianP50oxygen pressure at half-saturationn50Hill's cooperativity coefficientSAXSsmall angle x-ray scatteringMWCMonod-Wyman-Changeux model. Hb of the common North American earthworm Lumbricus terrestris is the most extensively studied of the annelid and vestimentiferan extracellular Hbs, giant (~3.5 MDa) heteromultimeric protein complexes of about 180 globin and nonglobin polypeptide chains that have a high cooperativity of oxygen binding and low iron and heme contents (1Vinogradov S.N. Lamy J. Truchot J.P. Gilles R. Respiratory Pigments in Animals. Springer-Verlag, Berlin1985: 9Crossref Google Scholar, 2Vinogradov S.N. Walz D.A. Pohajdak B. Moens L. Kapp O.H. Suzuki T. Trotman C.N.A. Comp. Biochem. Physiol. 1993; 106: 1-26Crossref Scopus (19) Google Scholar, 3Vinogradov S.N. Sharma P.K. Methods Enzymol. 1994; 231: 112-124Crossref PubMed Scopus (33) Google Scholar). The Lumbricus Hb is comprised of globin subunits T, a disulfide-bonded trimer of chains a-c (4Shishikura F. Mainwaring M.G. Yurewicz E.C. Lightbody J.L. Walz D.A. Vinogradov S.N. Biochim. Biophys. Acta. 1986; 869: 314-321Crossref PubMed Scopus (21) Google Scholar, 5Fushitani K. Matsuura M.S.A. Riggs A.F J. Biol. Chem. 1988; 263: 6502-6517Abstract Full Text PDF PubMed Google Scholar) and the monomer M (chain d) (6Vinogradov S.N. Shlom J.M. Hall B.C. Kapp O.H. Mizukami H. Biochim. Biophys. Acta. 1977; 492: 136-155Crossref PubMed Scopus (73) Google Scholar, 7Shishikura F. Snow J.W. Gotoh T. Vinogradov S.N. Walz D.A. J. Biol. Chem. 1987; 262: 3123-3131Abstract Full Text PDF PubMed Google Scholar), and four different types of linker chains of 24-32 kDa (8Suzuki T. Riggs A.F. J. Biol. Chem. 1993; 268: 13548-13555Abstract Full Text PDF PubMed Google Scholar, 9Martin P.D. Kuchumov A.R. Green B.R. Oliver R.W.A. Braswell E.H. Wall J.S. Vinogradov S.N. J. Mol. Biol. 1996; 255: 154-169Crossref PubMed Scopus (94) Google Scholar). Dissociation of the oxyHb at neutral pH in the presence of urea, Gdm salts, and other chaotropes at concentrations that do not denature the subunits, provides a 213-kDa dodecamer complex, consisting of T and M subunits (10Vinogradov S.N. Lugo S. Mainwaring M.G. Kapp O.H. Crewe A.V. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8034-8038Crossref PubMed Scopus (91) Google Scholar, 11Vinogradov S.N. Sharma P.K. Qabar A.N. Wall J.S. Westrick J.A. Simmons J.H. Gill S.J. J. Biol. Chem. 1991; 266: 13091-13096Abstract Full Text PDF PubMed Google Scholar). It has been shown recently that the dodecamer plays an important role in the dissociation and reassociation of the HBL structure (12Sharma P.K. Kuchumov A.R. Chottard G. Martin P.D. Wall J.S. Vinogradov S.N. J. Biol. Chem. 1996; 271: 8754-8762Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Furthermore, a preliminary x-ray diffraction study of oxydodecamer crystals has shown that it has an almost planar conformation (13Martin P.D. Eisele K.L. Doyle M.A. Kuchumov A.R. Walz D.A. Arutyunyan E.G. Vinogradov S.N. Edwards B.F.P. J. Mol. Biol. 1996; 255: 170-175Crossref PubMed Scopus (26) Google Scholar). Here we report the results obtained regarding the molecular shape and physical homogeneity of the oxydodecamer in solution at neutral pH, its dissociation, and its equilibrium oxygen binding. hexagonal bilayer hemoglobin guanidinium chloride potassium undecatungstosilicate sodium tetra-contatungstotetraarsenate(III) 2-bis(2-hydroxyethyl)-amino-2-(hydroxymethyl)-1,3-propanediol fast protein liquid chromatography absorbance unit root mean square exponentially modified gaussian oxygen pressure at half-saturation Hill's cooperativity coefficient small angle x-ray scattering Monod-Wyman-Changeux model. Materials—L. terrestris Hb was prepared as described previously in 0.1 M Tris·Cl buffer, pH 7.0, 1 mM EDTA, ~2 mM phenylmethanesulfonyl fluoride from live worms from around London, Ontario (Carolina Wholesale Bait Co., Canton, NC) (3Vinogradov S.N. Sharma P.K. Methods Enzymol. 1994; 231: 112-124Crossref PubMed Scopus (33) Google Scholar). Blood was the supernatant from the first centrifugation to remove cellular debris. The dodecamer was isolated by preparative gel filtration at neutral pH, subsequent to exposure to 4 M urea (11Vinogradov S.N. Sharma P.K. Qabar A.N. Wall J.S. Westrick J.A. Simmons J.H. Gill S.J. J. Biol. Chem. 1991; 266: 13091-13096Abstract Full Text PDF PubMed Google Scholar) or 10-30 mM SiW or AsW at 7°C; the fractions were screened by SDS-polyacrylamide gel electrophoresis (14Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and were pooled on the basis of their subunit content. The concentration was calculated from the absorbance of the cyano-met form at 540 nm using the extinction coefficient 0.656 ± 0.011 ml·mg−1·cm−1 for the monomer subunit (6Vinogradov S.N. Shlom J.M. Hall B.C. Kapp O.H. Mizukami H. Biochim. Biophys. Acta. 1977; 492: 136-155Crossref PubMed Scopus (73) Google Scholar). GdmCl was purum grade from Fluka AG (9470 Buchs, Switzerland), and urea was from Sigma. The heteropolytungstates SiW and AsW were synthesized as described by Klemperer (15Klemperer W.G. Inorganic Syntheses. Vol 27. John Wiley & Sons, Inc., New York1990: 71Google Scholar). Scattering experiments were performed using a Kratky compact camera with slit collimation, a position-sensitive detector (MBraun PSD-50M), and a Philips PW2253/11 x-ray tube with a copper target, operated at 50 kV and 30 mA. Sample solutions were placed in a 1-mm diameter Mark capillary and kept at 4°C during the measurements. A 30-µm nickel filter was employed to eliminate polychromatic effects. Using a sample to detector distance of about 20.9-cm, the width per detector channel corresponded to 0.00964 nm−1 on the h scale (h = (4π/λ)sinθ; 2θ is the scattering angle, λ = 0.154 nm is the wavelength of the CuKα line). Significant scattering data were collected in 300 channels corresponding to h values ranging from 0.056 to 2.0 nm−1. The counting time was typically 1800 s, and each scattering curve was recorded several times in order to reduce the statistical errors. Two series of measurements for the oxy and deoxy forms of the dodecamer were performed using five different concentrations ranging from 5 to 53 mg/ml in 0.1 M Tris·Cl buffer, pH 7.0, 1 mM EDTA. Data evaluation, including smoothing, desmearing, and indirect Fourier transformation, was carried out with the computer program ITP. Details of the experimental technique and the evaluation procedure are described elsewhere (16Pilz I. Glatter O. Kratky O. Methods Enzymol. 1979; 61: 148-249Crossref PubMed Scopus (99) Google Scholar, 17Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 1Google Scholar). A finite element method (small identical spheres) was used for model calculations to simulate the experimentally obtained scattering curves and distance distribution functions (18Glatter O. Acta Phys. Austriaca. 1980; 52: 243-250Google Scholar). Deoxygenation of the oxydodecamer solution was performed directly in the Mark capillary by repeated evacuation and equilibration with purified nitrogen. The spectral alterations attendant upon the conversion of oxy to deoxy forms, namely the disappearance of the two peaks at 540 and 575 nm and the appearance of a broad peak at 550 nm, were followed via the measurement of visible absorption spectra over the 490-650-nm region using the Mark capillary as the sample cell in a specially constructed device equipped with two adjustable slits and an attenuation filter as reference, placed in a Zeiss PMQII spectrophotometer. The reversibility of the oxy to deoxy spectral alteration was checked by reoxygenation of the sample in the capillary in the presence of air. For velocity sedimentation studies, 3- and 12-mm optical path double sector analytical ultracentrifuge cells were filled with oxydodecamer solutions (14 to 0.04 mg/ml) in 0.1 M Tris·Cl buffer, 1 mM EDTA, pH 7.0, using the dialysate for dilution of the stock solutions. The cell thickness (3 or 12 mm) and the wavelength of the light used to detect the gradient (280, 310, or 412 nm) were chosen so that the initial absorbance was ~2 for the high and ~0.1 for the low concentration samples. The appropriate dialysate was loaded in the reference side of each cell. The cells were centrifuged at 1, 25, and 40°C at 55,000 rpm in a Beckmann XL-A analytical ultracentrifuge, and the absorbance was measured radially for several hours until the boundaries had sedimented almost to the bottom of the cell. For sedimentation equilibrium experiments, oxydodecamer solutions were diluted using the dialysate to provide concentrations of 2, 1, 0.5, 0.25, 0.08, and 0.04 mg/ml. Approximately 25 or 100 µl were loaded into the sample channels of 6-channel sedimentation equilibrium cells (19Yphantis D.A. Ann. N. Y. Acad. Sci. 1960; 88: 586-601Crossref PubMed Scopus (326) Google Scholar, 20Ansevin A.T. Roark D.E. Yphantis D.A. Anal. Biochem. 1970; 34: 237-261Crossref PubMed Scopus (95) Google Scholar) with 3- and 12-mm optical paths and 3 and 10 µl of high density fluorocarbon oil (M&M FC-43) added, respectively, in order to raise the bottom of the solution and provide column heights of 2 mm in all cases. The solvent channels were filled with about 30 and 110 µl of dialysate, respectively. The concentration gradients were measured from the absorbance at 280, 418, and 540 nm, depending on the protein concentration. The cells were centrifuged in a Beckman model XL-A analytical ultracentrifuge at 14,000 rpm at 1°C for dodecamer from urea and 10,000 rpm at 1 and 25°C for dodecamer from SiW dissociation, respectively. Absorbance data were taken radially at 0.001-cm intervals and scans made every 3 h. Sedimentation equilibrium was determined to have been reached when successive scans were not detectably different as determined by a program (MATCH developed by D. Yphantis). Data sets were also obtained upon reaching equilibrium at 19,000 and 24,000 rpm for dodecamer from urea dissociation and 20,000 rpm for dodecamer from SiW dissociation. This range of speeds was necessary to resolve species from monomer to dodecamer. A nonlinear least squares program (21Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar) was used for data analysis. Low pressure, isocratic gel filtration was carried out at room temperature (20 ± 2°C) employing an FPLC system (Pharmacia Biotech Inc.) and 1 × 30-cm columns of Superose S12 or S6 (Pharmacia). Flow rate was 0.4 ml/min, and the eluate was monitored at 280 nm. A constant amount of protein in a constant sample volume, ~800 µg/200 µl, was loaded each time. The FPLC elution curves were acquired using the Easyest System 8 (Keithley Instruments, Inc. Rochester, NY) and an IBM PC386 computer and fitted with EMG's representing the undissociated dodecamer D, the trimer T, and the monomer subunit M, employing least squares minimization (Peak Fit Version 2.0, Jandel Scientific). The EMG function is a convolution of gaussian and decreasing exponential +functions, f(x)=aOexpa222a32+(a1−x)a31+erf22(x−a1a2−a2a3(Eq. 1) where a0 is the amplitude, a1 is the center, a2 is the width of the gaussian, and a3 is the width of the exponential. The EMG is asymmetric with an exponential tail on the right side, whose fall-off rate is controlled by the parameter a3. The EMG represents well the shape of chromatographic elution peaks (22Jonsson J.A. Chromatographic Theory and Basic Principles. Marcel Dekker Inc., New York1987Google Scholar, 23Reh E. Trends Anal. Chem. 1995; 14: 1-5Crossref Scopus (13) Google Scholar). Urea, GdmCl, or SiW was dissolved in 0.1 M Tris·Cl buffer, pH 7.0, 1 mM EDTA, and stock solution of the oxydodecamer added to obtain the desired concentration (~1.0 mg/ml). Conversion of oxy- to metdodecamer was affected by the addition of potassium ferricyanide (Fisher) or of sodium nitrite (Aldrich) at molar ratios relative to heme, ranging from 1 to 1000. Upon completion of the spectral alterations over the 450-650-nm range the oxidizing agent was removed by gel filtration on a 1.5 × 20-cm column of Sephadex G-25. The dissociations at neutral pH and at pH 8.0 were followed by FPLC. The areas of the individual peaks representing undissociated dodecamer D, trimer T, and monomer M expressed as percent of total were plotted versus time and fitted to sums of two exponentials, f(t)=a1exp[−k1t]+a2exp[−k2t](Eq. 2) employing PSI-Plot software (Poly Software Int., Salt Lake City, UT) and the Marquardt-Levenberg method. The fit was judged acceptable only in the absence of systematic trends in the plot of residuals with time. O2 binding equilibria were measured at 25 and 10°C, using a modified gas diffusion chamber allowing stepwise increases in O2 pressure (24Weber R.E. Nature. 1981; 292: 386-387Crossref Scopus (110) Google Scholar, 25Weber R.E. Jensen F.B. Cox R.P. J. Comp. Physiol. B Metab. Transp. Funct. 1987; 157: 145-152Crossref PubMed Scopus (61) Google Scholar). Variation in pH was obtained by adding Tris·Cl buffers of different pH. The chamber was coupled to cascaded Woesthoff (types M301, M201 and M101a-f) gas pumps for mixing pure N2 (99.998%), atmospheric air, and O2. The P50 and n50 were interpolated from linear plots of log(S/(1 − S)) versus log PO2 for S (fractional saturation) between 0.3 and 0.7. The O2 equilibrium measurements at extremes of saturation were measured as described previously (26Weber R.E. J. Appl. Physiol. 1992; 72: 1611-1615Crossref PubMed Scopus (97) Google Scholar, 27Weber R.E. Malte H. Braswell E.H. Oliver R.W.A. Green B.N. Sharma P.K. Kuchumov A. Vinogradov S.N. J. Mol. Biol. 1995; 251: 703-720Crossref PubMed Scopus (47) Google Scholar). Errors resulting from possible incomplete saturation in the presence of pure O2 were minimized by upper end point extrapolation of log(A) versus 1/PO2 plots to 1/PO2 = 0. Lower end point correction was carried out applying an A versus PO2 plot. The extrapolations were performed by fitting a cubic polynomial. The data were analyzed in terms of the two-state MWC equation (28Monod J. Wyman J. Changeux J.P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6184) Google Scholar): S=LKTP{1+KTP}(q−1)+KTP{1+KRP}(q−1)L(1+KTP)q+(1+KRP)q(Eq. 3) where S denotes saturation, P the partial pressure of O2, L the allosteric constant, KT and KR the association equilibrium constants for the low affinity (T, tense) and high affinity (R, relaxed) forms, respectively, and q the number of interacting binding sites. Nonlinear least squares fitting of the data in the form log(S/(1 − S)) versus log(P) was performed using the software package Mathematica (Wolfram Research Inc., Champaign, IL 61820) employing the Marquardt-Levenberg method. The final scattering curves I(h) for the oxy and deoxy forms of dodecamer from Hb dissociation in urea, obtained after extrapolation to zero concentration, are shown in Fig. 1A and the corresponding pair distance distribution functions p(r) in Fig. 1B. The molecular parameters derived from the two functions are provided in Table I; they appear to be identical within the error limits.Table I.SAXS molecular parameters for dodecamer and modelParameterDodecameraFrom Hb dissociation in urea.ModelbConsensus model (Fig. 2) based on averaging 33 well-fitting models using 265 overlapping spheres, 0.661 nm in diameter.OxyDeoxyRadius of gyration (nm)3.74 ± 0.013.73 ± 0.013.74 ± 0.05Maximum diameter (nm)10.59 ± 0.0110.59 ± 0.0110.7 ± 0.1Volume (nm3)255 ± 10250 ± 10249 ± 2Molecular mass (kDa)cBased on a calculated specific volume of 0.733.190 ± 19191 ± 19a From Hb dissociation in urea.b Consensus model (Fig. 2) based on averaging 33 well-fitting models using 265 overlapping spheres, 0.661 nm in diameter.c Based on a calculated specific volume of 0.733. Open table in a new tab The scattering curves and p(r) functions of a large number of models were calculated using small overlapping identical spheres to represent the protein mass (17Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 1Google Scholar) and compared with the experimental results. Only a few models were found to exhibit scattering equivalent to the dodecamer, i.e. fulfilling the following requirements within the mean square deviation, 1) same radius of gyration, 2) same scattering curve up to h = 1.9 nm−1 (corresponding to about 3 orders of magnitude in intensity), 3) same maximum diameter and shape of p(r) functions, and 4) similar volume. Fig. 2A shows the consensus model obtained by averaging 33 slightly different models, all of them fulfilling the above requirements. As a consequence of averaging, the spheres comprising the consensus model (265 spheres, radius 0.661 nm) are no longer all of equal weight. In the figure, the filled spheres have a high weight (i.e. common to at least 32 models), the empty spheres have a low weight (common to only one or two models), and the gray spheres have an intermediate weight. If this weighting is taken into account the I(h) and p(r) functions of the consensus model provide excellent agreement with the experimental curves (Fig. 3). Its x, y, and z dimensions are 9.6 ± 0.2, 8.5 ± 0.1, and 4.9 ± 0.3 nm, respectively; the molecular parameters are given in Table I.Fig. 3Comparison of the experimental scattering curve I(h) (A) and the pair distance distribution function p(r) of the oxydodecamer from Hb dissociation in urea (filled squares) with the curves calculated for the consensus model (solid line) and for the planar model (dashed line) in Fig. 2 (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Attempts to fit the experimental data with more oblate models were unsuccessful. Fig. 2B shows a planar model (223 spheres, radius 0.661 nm−1) which had the following molecular parameters, radius of gyration 3.75 nm, maximum dimension 10.8 nm, volume 270 nm3, dimensions x = y = 9.5 nm, and dimension z = 4.2 nm. The scattering curve of this model deviates from the experimental one (Fig. 3A) in the angular range h ≥1 nm−1), whereas the model shown in Fig. 2A fits the experimental curve up to h = 1.9 nm−1. Comparison of the pair distance distribution functions in Fig. 3B also shows considerable discrepancies in the case of the planar model. As a consequence of planarity, the shape of the p(r) function is changed in a way that the frequency of distances representing the “thickness” of the model is increased, leading to a shift of the p(r) function maximum to the left. The scans were analyzed by the dc/dt method (29Stafford W.F. Anal. Biochem. 1992; 203: 295-301Crossref PubMed Scopus (523) Google Scholar) to provide weight distributions of sedimentation coefficients (s), called g*s. Fig. 4 shows the results obtained at 1°C for the oxydodecamer from Hb dissociation in urea, in the absence and presence of 10 mM Ca2+. Fig. 5 shows the results obtained at 1, 25, and 40°C for the oxydodecamer prepared by Hb dissociation in SiW. The graphs were normalized to approximately equal areas in order to facilitate comparison of the two concentrations. The values of s corrected to standard conditions, i.e. s20w, are given in parentheses in the figure and in Table II.Fig. 5Sedimentation velocity of the oxydodecamer (4 and 0.04 mg/ml) prepared by Hb dissociation in SiW, in 0.1 M Tris·Cl buffer, pH 7.0, 1 mM EDTA, at 1°C (A), 25°C (B), and 40°C (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table II.Sw,20 from sedimentation velocity analysisDodecamer from urea dissociationDodecamer from SiW dissociationConcentrationCa2+ absentCa2+ presentConcentration1°C25°C40°CHigha12.4 and 13.5 mg/ml for Ca2+ absent and present, respectively.9.388.524.0 mg/ml9.188.5810.04.413.623.813.65Lowb0.039 and 0.041 mg/ml for Ca2+ absent and present, respectively.3.793.620.4 mg/ml9.448.221.891.893.634.003.48a 12.4 and 13.5 mg/ml for Ca2+ absent and present, respectively.b 0.039 and 0.041 mg/ml for Ca2+ absent and present, respectively. Open table in a new tab The experiments with oxydodecamer from urea dissociation produced 1818 data points in the absence and 1568 points in the presence of Ca2+ at 1°C. Global fits using the NONLIN program (21Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar) were obtained by assuming various models of self-association. The program calculates the value of the reduced molecular weight (M′ = M{1 − vρ}, where v is the specific volume, M is the molecular weight of the molecule, and ρ is the density of the solution), the values of other constants related to the model, i.e. second virial coefficient, equilibrium constants, etc., and the value of the r.m.s. error. M′ was converted to molecular weight using v = 0.733, calculated from the amino acid compositions (30Cohn E.J. Edsall J.T. Proteins, Amino Acids and Proteins. Reinhold Publishing Corp., New York1943: 370Google Scholar) of the globin subunits of Lumbricus Hb (5Fushitani K. Matsuura M.S.A. Riggs A.F J. Biol. Chem. 1988; 263: 6502-6517Abstract Full Text PDF PubMed Google Scholar, 7Shishikura F. Snow J.W. Gotoh T. Vinogradov S.N. Walz D.A. J. Biol. Chem. 1987; 262: 3123-3131Abstract Full Text PDF PubMed Google Scholar) and solvent densities estimated from density tables. The monomer-trimer and monomer-tetramer models gave the best fits; the results are provided in Table III. The experiments with oxydodecamer from SiW dissociation produced 12 sets of data consisting of about 1300 data points at both 1 and 25°C; the results of global fits with the NONLIN program are shown in Table III and the residuals of the fits in Fig. 6. Although the figure shows little systematic error for the two best fits, the monomer-tetramer model provided the best fit based on r.m.s. error, with ln K4 (association K4 in liters3/g3) being 7 at 1°C and 4 at 25°C.Table III.Monomer and n-mer masses from sedimentation equilibrium analysisModelDodecamer from urea dissociationDodecamer from SiW dissociationCa2+ absentCa2+ present1°C25°CMonn-merr.m.s.Monn-merr.m.s.Monn-merr.m.s.aRoot mean square error in 10−2 absorbance units.Monn-merr.m.s.Monomer-trimer58 ± 3174 ± 90.7251 ± 4153 ± 120.9964 ± 1192 ± 30.8366 ± 0.4198 ± 11.0Monomer-tetramer55 ± 4220 ± 160.6846 ± 5184 ± 200.9452 ± 1209 ± 60.7252 ± 0.5207 ± 20.6a Root mean square error in 10−2 absorbance units. Open table in a new tab Fig. 7A shows a typical FPLC elution profile at 280 nm of partially dissociated dodecamer and illustrates the fitting of the three peaks with EMG functions. The elution volumes of the peaks were unchanged during the course of the dissociation, the standard deviation of constant a1 in the EMG fit being <2%. The time course of oxydodecamer dissociation in 1.75 molal urea is shown in Fig. 7, B-D. The points represent averages of two separate experiments. The dissociation of the dodecamer was followed to >80% completion, ~1400 h. The insets show the initial 10% of the processes. In all cases, the data could be properly fitted only with two exponentials. The resulting residuals are shown at the bottom of the plots. Table IV provides the amplitudes and kinetic constants obtained from the fits. The presence of 10 mM Ca2+ did not have much effect on the dissociation of the oxydodecamer in urea.Table IV.Exponential fits for the dissociation of oxydodecamerConditionsa1k1× 10−5t1/2at1/2 = 0.693/K.a2k2× 10−5t1/2at1/2 = 0.693/K.rbThe correlation coefficient r = ΣXY/{(ΣX2) (ΣY2)}1/2, where X = xi− x and Y = yi− y.h−1hh−1h1.75 molal urea71360190251546000.9861.22 molal Gdm ·; Cl63660105221741000.989pH 8.049420017371205800.99110 mM SiW1236311000.996Metdodecamer755400130.986a t1/2 = 0.693/K.b The correlation coefficient r = ΣXY/{(ΣX2) (ΣY2)}1/2, where X = xi− x and Y = yi− y. Open table in a new tab Fig. 8 shows the time courses of oxydodecamer dissociation in 1.22 molal GdmCl at pH 7 (A), at pH 8.0 (B), in 10 mM SiW (C), and upon conversion to the met-form (D) at pH 7, together with the exponential fits and the resulting residuals. Although two exponentials are necessary to provide a reasonable fit to the dissociations in GdmCl and at pH 8, the dissociations in SiW and upon oxidation can be fitted with only a single exponential. The parameters obtained from the fit are given in Table IV. The parameters for Lumbricus blood, Hb, and the dodecamer from Hb dissociation in urea, in the absence and presence of Mg2+ and Ca2+, are shown in Fig. 9, Fig. 10 and Table V; the extended Hill plots are presented in Fig. 11 and the calculated MWC parameters in Table VI. The parameters for dode-camer from SiW dissociation are also provided in Table V., Table VI..Fig. 10Effect of Ca2+ and Mg2+ cations on P50 and n50 values of dodecamer from urea dissociation, Hb, and blood at 25°C. Empty column, at pH 7.86 in the absence of cations; solid column, at pH 7.18 in the absence of cations; hatched column, at pH 7.18 in the presence of 100 mM Mg2+; cross-hatched column, at pH 7.18 in the presence of 100 mM Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table V.Oxygenation parameters of Lumbricus blood, Hb, and dodecamerParametersP50 (torr)φaBohr coefficient = ΔlogK50/ΔpH.n50ΔH (kJ/mol)pH7.57.2-7.87.27.87.5Temp°C102525252510-25Blood3.28.5−0.425.29.5−45+100 mM Mg2+3.7−0.627.0+100 mM Ca2+2.9−0.536.0Hb4.011.5−0.244.54.9−50+100 mM Mg2+4.3−0.565.8+100 mM Ca2+3.4−0.495.7DodecamerbFrom Hb dissociation in 4 M urea.4.113.5−0.191.82.2−56+100 mM Mg2+5.2−0.612.6+100 mM Ca2+4.3−0.462.0Dodecamer bFrom Hb dissociation in 4 M urea., cIn 0.1 M BisTris/propane, pH 7.1, 0.1 M NaCl, 1 mM EDTA (11).11.72.1Hb11.9−0.173.2DodecamerdFrom Hb dissociation in SiW.11.9−0.162.7DodecamereFrom Hb dissociation in AsW.12.3−0.152.1a Bohr coefficient = ΔlogK50/ΔpH.b From Hb dissociation in 4 M urea.c In 0.1 M BisTris/propane, pH 7.1, 0.1 M NaCl, 1 mM EDTA (11Vinogradov S.N. Sharma P.K. Qabar A.N. Wall J.S. Westrick J.A. Simmons J.H. Gill S.J. J. Biol. Chem. 1991; 266: 13091-13096Abstract Full Text PDF PubMed Google Scholar).d From Hb dissociation in SiW.e From Hb dissociation in AsW." @default.
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