FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1964919153> ?p ?o ?g. }

• W1964919153 endingPage "20506" @default.
• W1964919153 startingPage "20499" @default.
• W1964919153 abstract "The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH4HCO3 buffer (pH 7.4, 20 mm, 25 °C) containing Zn2+ (100 μm), 14% of the heme dissociates from this complex (4.5 μm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO3 solution, and is minimal in NaHCO3. In NH4HCO3 buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn2+ > Cu2+ ≫ Ni2+ > Co2+≫Mn2+. Addition of Ca2+ prior to addition of Zn2+ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca2+ after Zn2+-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 °C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor. The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH4HCO3 buffer (pH 7.4, 20 mm, 25 °C) containing Zn2+ (100 μm), 14% of the heme dissociates from this complex (4.5 μm) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO3 solution, and is minimal in NaHCO3. In NH4HCO3 buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order Zn2+ > Cu2+ ≫ Ni2+ > Co2+≫Mn2+. Addition of Ca2+ prior to addition of Zn2+ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca2+ after Zn2+-induced dissociation of the Hx-heme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37 °C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor. The plasma protein hemopexin (Hx, 2The abbreviations used are: HxhemopexinMWmolecular weight. MW ∼58,000) is a positive acute phase reactant glycoprotein with the primary role of scavenging heme released from methemoglobin and other heme proteins as the result of hemolysis, rhabdomyolysis, or ischemia-reperfusion injury (1Delanghe J.R. Langlois M.R. Clin. Chim. Acta. 2001; 312: 13-23Crossref PubMed Scopus (194) Google Scholar, 2Tolosano E. Altruda F. DNA Cell Biol. 2002; 21: 297-306Crossref PubMed Scopus (323) Google Scholar, 3Tolosano E. Fagoonee S. Morello N. Vinchi F. Fiorito V. Antioxid. Redox. Signal. 2010; 12: 305-320Crossref PubMed Scopus (201) Google Scholar). In doing so, Hx prevents the oxidative damage and pro-inflammatory effects of free heme (4Tolosano E. Hirsch E. Patrucco E. Camaschella C. Navone R. Silengo L. Altruda F. Blood. 1999; 94: 3906-3914Crossref PubMed Google Scholar, 5Holt S. Reeder B. Wilson M. Harvey S. Morrow J.D. Roberts 2nd, L.J. Moore K. Lancet. 1999; 353: 1241Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 6Jeney V. Balla J. Yachie A. Varga Z. Vercellotti G.M. Eaton J.W. Balla G. Blood. 2002; 100: 879-887Crossref PubMed Scopus (515) Google Scholar) as demonstrated by the increased sensitivity of Hx-null mice (4Tolosano E. Hirsch E. Patrucco E. Camaschella C. Navone R. Silengo L. Altruda F. Blood. 1999; 94: 3906-3914Crossref PubMed Google Scholar, 7Tolosano E. Fagoonee S. Hirsch E. Berger F.G. Baumann H. Silengo L. Altruda F. Blood. 2002; 100: 4201-4208Crossref PubMed Scopus (116) Google Scholar) to heme overload and development of heme-catalyzed oxidative damage to the vasculature, liver, and kidneys. Hx may also have a role in protection of neural tissue, and it exhibits other activities that include inhibition of necrosis and adhesion of polymorphonuclear leukocytes (8Tolosano E. Cutufia M.A. Hirsch E. Silengo L. Altruda F. Biochem. Biophys. Res. Commun. 1996; 218: 694-703Crossref PubMed Scopus (35) Google Scholar, 9Suzuki K. Kobayashi N. Doi T. Hijikata T. Machida I. Namiki H. Cell Struct. Funct. 2003; 28: 243-253Crossref PubMed Scopus (10) Google Scholar, 10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar). The dominant form of circulating Hx is the apoprotein 3ApoHx refers to the hemopexin glycoprotein without ferriprotoporphyrin IX bound at a site equivalent to that identified in the rabbit Hx structure (21) and implies nothing concerning the binding of metal ions. (6–25 μm (1Delanghe J.R. Langlois M.R. Clin. Chim. Acta. 2001; 312: 13-23Crossref PubMed Scopus (194) Google Scholar)), which upon binding heme forms the Hx-heme complex that is recognized by hepatic receptors and removed from circulation by endocytosis (3Tolosano E. Fagoonee S. Morello N. Vinchi F. Fiorito V. Antioxid. Redox. Signal. 2010; 12: 305-320Crossref PubMed Scopus (201) Google Scholar, 11Hvidberg V. Maniecki M.B. Jacobsen C. Højrup P. Møller H.J. Moestrup S.K. Blood. 2005; 106: 2572-2579Crossref PubMed Scopus (350) Google Scholar, 12Smith A. Rish K.R. Lovelace R. Hackney J.F. Helston R.M. Biometals. 2009; 22: 421-437Crossref PubMed Scopus (9) Google Scholar). Subsequent to endocytosis, heme is dissociated from the Hx-heme complex and oxidized by heme oxygenase-1 to release iron that is then added to ferritin stores (13Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar, 14Davies D.M. Smith A. Muller-Eberhard U. Morgan W.T. Biochem. Biophys. Res. Commun. 1979; 91: 1504-1511Crossref PubMed Scopus (45) Google Scholar). The protein component of the endocytosed Hx-heme complex that is liberated in this manner either returns to circulation (15Smith A. Morgan W.T. Biochem. J. 1979; 182: 47-54Crossref PubMed Scopus (132) Google Scholar, 16Smith A. Hunt R.C. Eur. J. Cell Biol. 1990; 53: 234-245PubMed Google Scholar, 17Okazaki H. Taketani S. Kohno H. Tokunaga R. Kobayashi Y. Cell Struct. Funct. 1989; 14: 129-140Crossref PubMed Scopus (17) Google Scholar, 18Potter D. Chroneos Z.C. Baynes J.W. Sinclair P.R. Gorman N. Liem H.H. Muller-Eberhard U. Thorpe S.R. Arch. Biochem. Biophys. 1993; 300: 98-104Crossref PubMed Scopus (20) Google Scholar) or undergoes lysosomal degradation (11Hvidberg V. Maniecki M.B. Jacobsen C. Højrup P. Møller H.J. Moestrup S.K. Blood. 2005; 106: 2572-2579Crossref PubMed Scopus (350) Google Scholar, 18Potter D. Chroneos Z.C. Baynes J.W. Sinclair P.R. Gorman N. Liem H.H. Muller-Eberhard U. Thorpe S.R. Arch. Biochem. Biophys. 1993; 300: 98-104Crossref PubMed Scopus (20) Google Scholar). hemopexin molecular weight. Heme binds to Hx with extremely high affinity (19Hrkal Z. Vodrázka Z. Kalousek I. Eur. J. Biochem. 1974; 43: 73-78Crossref PubMed Scopus (170) Google Scholar, 20Miller Y.I. Shaklai N. Biochim. Biophys. Acta. 1999; 1454: 153-164Crossref PubMed Scopus (120) Google Scholar) even though the crystal structure for the rabbit Hx-heme complex indicates that the heme is more exposed to solvent than is the case for many heme proteins (21Paoli M. Anderson B.F. Baker H.M. Morgan W.T. Smith A. Baker E.N. Nat. Struct. Biol. 1999; 6: 926-931Crossref PubMed Scopus (207) Google Scholar, 22Morgan W.T. Sutor R.P. Muller-Eberhard U. Biochim. Biophys. Acta. 1976; 434: 311-323Crossref PubMed Scopus (23) Google Scholar). The Hx-heme complex is an unusual b-type hemeprotein in that the axial ligands that coordinate the heme iron are not located in a structurally constrained region of the protein, and one of the axial histidine ligands is located in the flexible linker region that connects the N- and C-terminal domains of the protein (10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar, 21Paoli M. Anderson B.F. Baker H.M. Morgan W.T. Smith A. Baker E.N. Nat. Struct. Biol. 1999; 6: 926-931Crossref PubMed Scopus (207) Google Scholar). Physiologically, Hx must bind heme with extremely high affinity so that it can scavenge heme in circulating blood under conditions where it is released by myoglobin and hemoglobin, but Hx must also release heme in hepatic endosomes potentially liberating apoHx to return to circulation. While the high affinity of Hx for heme may be attributed to bisimidazole axial coordination, to packing of hydrophobic residues around the heme, and to the hydrogen bonding interactions of the heme propionate groups (21Paoli M. Anderson B.F. Baker H.M. Morgan W.T. Smith A. Baker E.N. Nat. Struct. Biol. 1999; 6: 926-931Crossref PubMed Scopus (207) Google Scholar, 23Deeb R.S. Muller-Eberhard U. Peyton D.H. Biochim. Biophys. Acta. 1994; 1200: 161-166Crossref PubMed Scopus (8) Google Scholar), structural characteristics of the protein that might promote the release of heme when required are less evident. Our recent studies of human Hx have focused on the effects of metal ions on the structural dynamics of this protein (24Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2007; 46: 9301-9309Crossref PubMed Scopus (9) Google Scholar), the thermal stability of the Hx-heme complex (10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar, 25Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2005; 44: 1872-1879Crossref PubMed Scopus (24) Google Scholar), and the influence of heme orientation on interaction of the protein with metal ions (26Mauk M.R. Rosell F.I. Mauk A.G. Biochemistry. 2007; 46: 15033-15041Crossref PubMed Scopus (8) Google Scholar). The reduction potential of the rabbit Hx-heme complex has been shown to be sensitive to the ionic composition of the solution and pH (27Flaherty M.M. Rish K.R. Smith A. Crumbliss A.L. Biometals. 2008; 21: 239-248Crossref PubMed Scopus (10) Google Scholar), and the human Hx-ferroheme complex exhibits greater thermal instability (lower Tm (25Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2005; 44: 1872-1879Crossref PubMed Scopus (24) Google Scholar, 28Shipulina N.V. Smith A. Morgan W.T. J. Protein Chem. 2001; 20: 145-154Crossref PubMed Scopus (12) Google Scholar)) than does the Hx-ferriheme complex. Nevertheless, the physiological significance of the many metal ion binding sites of Hx (10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar, 29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar) and of heme insertion isoforms exhibited by the Hx-heme complex remain uncertain. Recently, we have reported the ability of metal ions to induce heme release from a variety of b-type heme proteins (30Mauk M.R. Rosell F.I. Mauk A.G. J. Am. Chem. Soc. 2009; 131: 16976-16983Crossref PubMed Scopus (8) Google Scholar) at alkaline pH in bicarbonate buffer. We have now extended these studies to consideration of the Hx-heme complex and find that the stability of this complex also responds to electrolytes and metal ions at physiological pH but in a manner that should promote preferential binding of heme to apoHx in plasma. The possible implications of these results for the physiological function of Hx are discussed. Human Hx was isolated from plasma cryosupernate (Canadian Blood Services) without heme bound (apoHx), and the Hx-ferriprotoporphyrin IX complex (Hx-heme) was prepared by addition of ferriheme (Frontier Scientific) as described previously (24Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2007; 46: 9301-9309Crossref PubMed Scopus (9) Google Scholar, 25Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2005; 44: 1872-1879Crossref PubMed Scopus (24) Google Scholar, 29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar). The electronic absorption and CD spectra of the protein used in the current study were consistent with Form β of Hx-heme (26Mauk M.R. Rosell F.I. Mauk A.G. Biochemistry. 2007; 46: 15033-15041Crossref PubMed Scopus (8) Google Scholar). The Hx-heme complex was exchanged (≥4 × 105-fold) into water by ultrafiltration (AmiconUltra 10,000 NMWL Millipore) prior to dilution (∼50-fold) into buffers used for data collection. Hx-heme complex concentrations were determined on the basis of a molar absorptivity of 136,000 m−1 cm−1 at 280 nm (29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar, 31Seery V.L. Hathaway G. Eberhard U.M. Arch. Biochem. Biophys. 1972; 150: 269-272Crossref PubMed Scopus (51) Google Scholar). Horse heart myoglobin (Mb; Sigma cat. no. M1882) was converted to metmyoglobin (metMb) with K3Fe(CN)6 and passed over a column of Dowex 1X-8 (32Linder R.E. Records R. Barth G. Bunnenberg E. Djerassi C. Hedlund B.E. Rosenberg A. Benson E.S. Seamans L. Moscowitz A. Anal. Biochem. 1978; 90: 474-480Crossref PubMed Scopus (14) Google Scholar). The concentration of metMb was determined from the molar absorptivity (188,000 m−1 cm−1) at 408 nm (pH 6.4) (33Antonini E. Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. North-Holland Publishing Co., London1971Google Scholar). Spectroscopy and kinetics experiments were conducted with Cary Models 6000 and 3 spectrophotometers. All samples were thermostatted in Teflon-stoppered cuvettes with 1 cm pathlength (Hellma). Dissociation of heme from the Hx-heme complex was monitored at the Soret maximum (413.5 nm). Complete dissociation of heme from its normal binding site resulted in a 69.2% decrease in absorbance intensity at this wavelength (pH 7.4, 25 °C). Titrations of the Hx-heme complex with Ca2+ were monitored by following the rate of heme release as a function of the amount of CaCl2 added and were fit by non-linear regression analysis (Scientist, vers. 2, MicroMath) as described previously (34Mauk M.R. Ferrer J.C. Mauk A.G. Biochemistry. 1994; 33: 12609-12614Crossref PubMed Scopus (86) Google Scholar). Near UV and visible CD spectra were recorded with a Jasco Model J-810 spectropolarimeter. Prior to monitoring the exchange of heme from metMb to apoHx, each protein was exchanged (>5 × 104-fold) into buffer by centrifugal ultrafiltration. The transfer of heme from metMb to apoHx was monitored at the wavelength at which the greatest change in absorbance is observed for this reaction (404.5 nm). Glass-distilled water passed through a Barnstead Nanopure Diamond Life Sciences purification system was used throughout. All buffers (Fisher and Sigma) and salt solutions were air-saturated at 22 °C prior to adjusting pH. Tetramethylammonium hydroxide pentahydrate was purchased from Sigma. Metal ion solutions were prepared either by dilution from Titrosol (E. Merck) standards (CoCl2, CuCl2, MnCl2, and ZnCl2) or gravimetrically from metal chloride or sulfate salts (Fisher and Sigma). SDS-PAGE was performed under reducing conditions (29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar) to check for protein hydrolysis using PageRuler (Fermentas Life Sciences) molecular weight standards. Gels were analyzed with a ChemiGenius2 Bio Imaging System and associated software (GeneSnap 6.05 for image acquisition and GeneTools 3.06 for data analysis, Syngene). The spectrum of the Hx-heme complex is initially the same in either sodium or ammonium bicarbonate buffer (Fig. 1, spectrum a). With addition of Zn2+ (100 μm), half of the heme dissociates from this complex (4.3 μm) after 20 h in the presence of sodium bicarbonate buffer (20 mm, pH 7.4, 22 °C) (Fig. 1, spectrum e) whereas just 2.5 h is required for half of the heme to dissociate from the complex in ammonium bicarbonate buffer (20 mm, pH 7.4, 22 °C)(Fig. 1, spectrum d) and >97% of the heme was released after 22 h under these conditions. In ammonium bicarbonate buffer without added metal ions, the spectrum changes slowly in a manner that is consistent with dissociation of some (13% in 24 h) of the heme. This change in spectrum was unaffected by the presence of EDTA. In the absence of added metal ions, the spectrum of the Hx-heme complex changed <1% in sodium bicarbonate buffer in 24 h. This behavior of the Hx-heme complex is in marked contrast to our previous observations with other b-type heme proteins (30Mauk M.R. Rosell F.I. Mauk A.G. J. Am. Chem. Soc. 2009; 131: 16976-16983Crossref PubMed Scopus (8) Google Scholar) for which the dissociation of heme from the protein-heme complex was observed only after the addition of metal ions and the stability of heme binding was much greater in ammonium bicarbonate buffer than in sodium bicarbonate. While heme dissociates from the Hx-heme complex in the absence of metal ions in ammonium bicarbonate buffer (20 mm, pH 7.8), complete dissociation of the complex was not achieved even after more than 2 weeks at 15 °C (Fig. 2B). Electronic absorption spectra exhibited the same isosbestic points in the Soret and visible regions (395, 491, 507, and 590 nm) for the reaction with or without metal ions present (FIGURE 1, FIGURE 2). The influence of monovalent cations on the stability of the Hx-heme complex was examined in bicarbonate buffer (20 mm, pH 8.0 ± 0.2) by monitoring the decrease in absorbance at the Soret band (Fig. 3). These data were collected at 15 °C to ensure the stability of any apoHx formed during these extended reaction times. While the stability of the Hx-heme complex was only slightly diminished in potassium relative to sodium bicarbonate, ammonium bicarbonate had a pronounced destabilizing effect. This effect could result from an increase in the rate of release of heme from the Hx-heme complex (off-rate) or a decrease in the rate at which heme rebinds to apoHx (on-rate) or some combination of both as the counter ion is changed from sodium to potassium to ammonium. The effects of divalent metal cations (100 μm) on the rate of heme dissociation from the Hx-heme complex (4.3 μm) in ammonium bicarbonate buffer (20 mm, pH 7.4, 25 °C) are shown in Fig. 4. Metal ion concentrations of 100 μm were selected to saturate the principal metal ion binding sites of the protein as determined from potentiometric titrations (29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar). The enhancement of heme dissociation from the Hx-heme complex in ammonium bicarbonate buffer by various metal ions decreased in the order Zn2+ > Cu2+ ≫ Ni2+ > Co2+. The kinetics of all of these reactions are multiphasic (FIGURE 3, FIGURE 4). In the presence of divalent metal ions, the rate constant for association of heme with Hx is likely to be diminished because the metal ions presumably compete with the heme iron for coordination to the histidyl residues that normally provide axial ligands (10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar). Subtle changes in protein structure lower the Tm for the Hx-heme complex in the presence of these four metal ions (24Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2007; 46: 9301-9309Crossref PubMed Scopus (9) Google Scholar, 25Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2005; 44: 1872-1879Crossref PubMed Scopus (24) Google Scholar). These metal ion-induced structural changes are also expected to influence the rate constants for both association and dissociation of the Hx-heme complex. Notably, the addition of Mn2+ had little or no effect on the release of heme from the Hx-heme complex (Fig. 4). Previous potentiometric studies of Mn2+ binding to apoHx (29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar) demonstrated the presence of a high affinity binding site for Mn2+ on apoHx that is not exhibited by the Hx-heme complex. Changing the counter ion of the added metal ions from Cl− (200 μm) to SO42− (100 μm) produced no significant differences in the reaction rates. However, we note that the concentration of counter ion introduced with the metal salts is small relative to the concentration of Cl− (∼1.7 mm) required to lower the pH of the bicarbonate solutions to 7.4. In contrast to the other metal ions evaluated in this work, Ca2+ did not induce the release of heme from the Hx-heme complex in bicarbonate buffers but promoted the rebinding of any heme that had been released from the Hx-heme complex in either bicarbonate buffer alone or in bicarbonate with other metal ions such as Zn2+ added (Fig. 5). This observation suggests that binding of Ca2+ to the Hx-heme complex stabilizes the complex and decreases the rate of complex dissociation. This stabilization of heme binding to Hx by Ca2+ is observed in both ammonium (Fig. 5A) and potassium bicarbonate (Fig. 5C) and at 37 °C (Fig. 5, B and C) as well as at 15 °C (Fig. 5A). The rebinding of heme at 15 °C is slow and is likely influenced by the aggregation state of the released heme (30Mauk M.R. Rosell F.I. Mauk A.G. J. Am. Chem. Soc. 2009; 131: 16976-16983Crossref PubMed Scopus (8) Google Scholar, 35Kuzelová K. Mrhalová M. Hrkal Z. Biochim. Biophys. Acta. 1997; 1336: 497-501Crossref PubMed Scopus (57) Google Scholar). Stabilization of heme binding to Hx by Ca2+ is consistent with the previous observation that Ca2+ increases the Tm for loss of tertiary structure and for release of heme from the Hx-heme complex in sodium phosphate buffer (pH 7.4, 10 mm) (10Mauk M.R. Rosell F.I. Mauk A.G. Nat. Prod. Rep. 2007; 24: 523-532Crossref PubMed Scopus (6) Google Scholar). The CD spectrum of the Hx-heme complex formed as the result of Ca2+-induced rebinding of heme (data not shown) indicates regeneration of Form β (26Mauk M.R. Rosell F.I. Mauk A.G. Biochemistry. 2007; 46: 15033-15041Crossref PubMed Scopus (8) Google Scholar), which was the original form of the Hx-heme complex used in this study. The effects of metal ions described here are not expected to depend on the orientation of heme binding within the Hx-heme complex because Forms α and β exhibit the same metal ion-induced decreases in Tm for thermally-induced dissociation of the Hx-heme complex (25Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2005; 44: 1872-1879Crossref PubMed Scopus (24) Google Scholar, 26Mauk M.R. Rosell F.I. Mauk A.G. Biochemistry. 2007; 46: 15033-15041Crossref PubMed Scopus (8) Google Scholar). The stoichiometry of Ca2+ binding was examined in potassium bicarbonate buffer (20 mm, pH 8.0, 37 °C) by monitoring the rate of heme release as a function of added Ca2+. The data were fitted (Fig. 6) to a two-site binding model (34Mauk M.R. Ferrer J.C. Mauk A.G. Biochemistry. 1994; 33: 12609-12614Crossref PubMed Scopus (86) Google Scholar). Although this fit suggests the occurrence of two binding sites with affinities that differ by ∼3-fold, the inherent uncertainty of the data that could be collected for this process was sufficiently great that a more quantitative numerical analysis was not possible. The influence of Ca2+ on the kinetics of heme transfer from metMb to apoHx was investigated in an initial effort to evaluate the effect of this metal ion on heme exchange between proteins (Fig. 7). This reaction was monitored at the wavelength expected to exhibit the greatest change in absorbance (404.5 nm) as determined from the Hx-heme complex minus metMb difference spectrum (Fig. 7, inset) under conditions similar to those used to evaluate the effect of Ca2+ on the stability of the Hx-heme complex (FIGURE 5, FIGURE 6). The concentration of Ca2+ used (10 μm) was based on the stoichiometry of Ca2+ binding (Fig. 6). No precipitation of either apoMb or apoHx was observed under these conditions. Notably, both the rate and the extent of heme transfer from met Mb to apoHx increase in the presence of Ca2+ (Fig. 7). The relative stability of the Hx-heme complex (15 °C, pH 7.9) in the presence of ammonium salts (∼20 mm) of several common anions is shown in Fig. 8. As can be seen, these anions stabilize heme binding to Hx in the order Cl− ≥ HPO42− > HCO3−. The NH4Cl (20 mm) and NH4H2PO4 (10 mm) solutions were adjusted to pH 7.9 ± 0.1 with NH4OH such that the final ammonium concentrations were 21.6 and 22.1 mm, respectively. The extent of stabilization by chloride was difficult to establish quantitatively because maintaining the pH of NH4Cl solutions under aerobic conditions is difficult, and additionally, the chief contaminant in NH4Cl is NaCl (36O'Neil M.J. Merck Index. Twelfth Ed. Merck & Co., White House Station2006Google Scholar). As noted above, Na+ has a pronounced stabilizing effect on the Hx-heme complex relative to NH4+. Further clarification of the effect of chloride was sought by examining the stability of the Hx-heme complex in tetramethylammonium chloride (tetramethyl ammonium hydroxide (20 mm) adjusted to pH 8 with HCl ([Cl−]final = ∼18.6 mm)). Under these conditions, the stability of heme binding to Hx was intermediate between that observed in the presence of ammonium chloride and ammonium phosphate during initial incubation at 15 °C (Fig. 8). However, after prolonged exposure (∼5 days) to tetramethylammonium chloride, the Hx-heme complex exhibited sufficient turbidity that further analysis was precluded. The stabilizing effects of various electrolytes described above appear to be additive such that sodium phosphate buffer stabilizes the Hx-heme complex more than any of the other buffer combinations examined (Fig. 9). Interestingly, the electronic absorption spectrum of Hx-heme samples diluted into sodium phosphate buffers exhibited an increase in intensity of up to ∼4% at the Soret maximum (413.5 nm) over 24–48 h, and no further change was observed following prolonged incubation in this buffer (Fig. 9A). We attribute this increase in absorbance to rebinding of the small amount of heme that can be released from the Hx-heme during extended buffer exchange and storage in water. Addition of metal ions to the Hx-heme complex also promotes dissociation of heme in phosphate buffers although the effect is much smaller than observed in bicarbonate buffers as shown for addition of Zn2+ in Fig. 9B. The relationship between the ability of metal ions to destabilize the Hx-heme complex and the buffer composition is complex. For example, in sodium phosphate buffer (Fig. 9C) the relative effectiveness of Zn and Cu in promoting heme release is the opposite of that observed in ammonium bicarbonate buffer (Fig. 3). The possibility of metal ion promoted hydrolysis of Hx during the kinetics experiments was evaluated by analysis of these samples by SDS-PAGE in the presence of dithiothreitol (data not shown). No cleavage of Hx (apparent MW ∼74,000 (29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar)) was observed even after exposure to metal ions for 2 weeks at 15 °C except for samples containing Ni2+ (100 μm with 4–6 μm Hx). Analysis of samples of the Hx-heme complex following incubation with Ni2+ at 15 °C detected no hydrolysis of the Hx-heme complex but did detect cleavage of apoHx that formed during incubation (∼30% of the apoHx present hydrolyzed each day). The products of the Ni2+-catalyzed reaction exhibit apparent MWs of 28 kDa (one band) and 46 kDa (two bands that differ by ∼2 kDa). This banding pattern does not change upon further incubation at 15 °C, indicating no further hydrolysis occurred. At temperatures >22 °C, cleavage of apoHx by Ni2+ was significantly diminished or undetectable, consistent with our previous observations (24Rosell F.I. Mauk M.R. Mauk A.G. Biochemistry. 2007; 46: 9301-9309Crossref PubMed Scopus (9) Google Scholar). Ni2+ has been reported recently to hydrolyze the peptide bond preceding the Thr residue in model peptides possessing several sequences, including the sequence Thr-His-His (37Krezel A. Mylonas M. Kopera E. Bal W. Acta Biochim. Pol. 2006; 53: 721-727Crossref PubMed Scopus (31) Google Scholar, 38Krezel A. Kopera E. Protas A.M. Poznański J. Wyslouch-Cieszyńska A. Bal W. J. Am. Chem. Soc. 2010; 132: 3355-3366Crossref PubMed Scopus (54) Google Scholar), which occurs in human Hx (residues 225–227). While the hydrolysis we observe occurred at 15 °C, suggesting a limited physiological importance, we interpret the products formed by exposure of Hx to Ni2+ to correspond to the C-terminal residues 225–439 (MWcalc = 23,772 plus 1 carbohydrate chain) and the N-terminal residues 1–224 (MWcalc = 25,542 plus 5 carbohydrate chains). We assign the 46 kDa band from the SDS-PAGE to the N-domain based on this larger than anticipated apparent MW as well as the electrophoretic heterogeneity of native Hx reported previously (26Mauk M.R. Rosell F.I. Mauk A.G. Biochemistry. 2007; 46: 15033-15041Crossref PubMed Scopus (8) Google Scholar, 29Mauk M.R. Rosell F.I. Lelj-Garolla B. Moore G.R. Mauk A.G. Biochemistry. 2005; 44: 1864-1871Crossref PubMed Scopus (30) Google Scholar, 39Kamboh M.I. Ferrell R.E. Am. J. Hum. Genet. 1987; 41: 645-653PubMed Google Scholar). Our fragment sizes also agree well with apparent molecular weights reported for the N- and C-domains generated by proteolytic hydrolysis (40Muller-Eberhard U. Methods Enzymol. 1988; 163: 536-565Crossref PubMed Scopus (8" @default.
• W1964919153 created "2016-06-24" @default.
• W1964919153 creator A5010109402 @default.
• W1964919153 creator A5091229812 @default.
• W1964919153 date "2010-07-01" @default.
• W1964919153 modified "2023-09-27" @default.
• W1964919153 title "Metal Ions and Electrolytes Regulate the Dissociation of Heme from Human Hemopexin at Physiological pH" @default.
• W1964919153 cites W1514851383 @default.
• W1964919153 cites W1522231032 @default.
• W1964919153 cites W1548493774 @default.
• W1964919153 cites W1581806572 @default.
• W1964919153 cites W1591678396 @default.
• W1964919153 cites W171379636 @default.
• W1964919153 cites W1915072125 @default.
• W1964919153 cites W1929497397 @default.
• W1964919153 cites W1965153009 @default.
• W1964919153 cites W1971861352 @default.
• W1964919153 cites W1981534284 @default.
• W1964919153 cites W1981728824 @default.
• W1964919153 cites W1983891046 @default.
• W1964919153 cites W1986921744 @default.
• W1964919153 cites W1993414947 @default.
• W1964919153 cites W1994190762 @default.
• W1964919153 cites W1995745202 @default.
• W1964919153 cites W2003244187 @default.
• W1964919153 cites W2005496979 @default.
• W1964919153 cites W2006828869 @default.
• W1964919153 cites W200833201 @default.
• W1964919153 cites W2009736111 @default.
• W1964919153 cites W2010133611 @default.
• W1964919153 cites W2011467527 @default.
• W1964919153 cites W2015399255 @default.
• W1964919153 cites W2035854223 @default.
• W1964919153 cites W2040506379 @default.
• W1964919153 cites W2040874701 @default.
• W1964919153 cites W2045192433 @default.
• W1964919153 cites W2049501307 @default.
• W1964919153 cites W2053359068 @default.
• W1964919153 cites W2056010015 @default.
• W1964919153 cites W2060610643 @default.
• W1964919153 cites W2063505569 @default.
• W1964919153 cites W2065284764 @default.
• W1964919153 cites W2068616327 @default.
• W1964919153 cites W2074063169 @default.
• W1964919153 cites W2075045260 @default.
• W1964919153 cites W2081212628 @default.
• W1964919153 cites W2084206407 @default.
• W1964919153 cites W2085584456 @default.
• W1964919153 cites W2096413727 @default.
• W1964919153 cites W2097097625 @default.
• W1964919153 cites W2106348878 @default.
• W1964919153 cites W2110119945 @default.
• W1964919153 cites W2111897470 @default.
• W1964919153 cites W2135224653 @default.
• W1964919153 cites W2146055699 @default.
• W1964919153 cites W2153410182 @default.
• W1964919153 cites W2169450299 @default.
• W1964919153 cites W2169952241 @default.
• W1964919153 cites W2174160083 @default.
• W1964919153 cites W2174721344 @default.
• W1964919153 cites W2407954187 @default.
• W1964919153 cites W4206078312 @default.
• W1964919153 cites W73673827 @default.
• W1964919153 cites W95401234 @default.
• W1964919153 doi "https://doi.org/10.1074/jbc.m110.123406" @default.
• W1964919153 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2898363" @default.
• W1964919153 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20430887" @default.
• W1964919153 hasPublicationYear "2010" @default.
• W1964919153 type Work @default.
• W1964919153 sameAs 1964919153 @default.
• W1964919153 citedByCount "3" @default.
• W1964919153 countsByYear W19649191532018 @default.
• W1964919153 crossrefType "journal-article" @default.
• W1964919153 hasAuthorship W1964919153A5010109402 @default.
• W1964919153 hasAuthorship W1964919153A5091229812 @default.
• W1964919153 hasBestOaLocation W19649191531 @default.
• W1964919153 hasConcept C102931765 @default.
• W1964919153 hasConcept C12554922 @default.
• W1964919153 hasConcept C145148216 @default.
• W1964919153 hasConcept C147789679 @default.
• W1964919153 hasConcept C17525397 @default.
• W1964919153 hasConcept C178790620 @default.
• W1964919153 hasConcept C179104552 @default.
• W1964919153 hasConcept C181199279 @default.
• W1964919153 hasConcept C185592680 @default.
• W1964919153 hasConcept C199164860 @default.
• W1964919153 hasConcept C2776217839 @default.
• W1964919153 hasConcept C2776474381 @default.
• W1964919153 hasConcept C544153396 @default.
• W1964919153 hasConcept C55493867 @default.
• W1964919153 hasConcept C68801617 @default.
• W1964919153 hasConcept C75473681 @default.
• W1964919153 hasConcept C86803240 @default.
• W1964919153 hasConceptScore W1964919153C102931765 @default.
• W1964919153 hasConceptScore W1964919153C12554922 @default.
• W1964919153 hasConceptScore W1964919153C145148216 @default.
• W1964919153 hasConceptScore W1964919153C147789679 @default.
• W1964919153 hasConceptScore W1964919153C17525397 @default.

• next