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J M Rifkind

Publications and source records attributed to J M Rifkind.

At least 19 recordsLinked to original sources
PubMed1992

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Autoxidation of hemoglobin enhanced by dissociation into dimers.

Autoxidation as a function of hemoglobin concentration indicates a 17-fold increase in the rate of autoxidation from 0.25 (%/h) to 4.3 (%/h) when tetrameric oxyhemoglobin dissociates into dimers. As a result of this large enhancement, a contribution of dissociation to the autoxidation is evident even at relatively high concentrations of hemoglobin for which it is usually considered that dissociation can be neglected. The mechanism for this phenomenon is attributed to alterations in the ligand pocket which occur when constraints due to subunit contacts within the R-state are eliminated.

Erythrocytes↗
PubMed1991

Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.

Binding Sites↗
PubMed1991

The hypoxic stress on erythrocytes associated with superoxide formation.

Superoxide is produced during the autoxidation of hemoglobin. Autoxidation of hemoglobin is, however, facilitated under hypoxic conditions where hemoglobin is only partially oxygenated. We have recently found that the erythrocyte superoxide dismutase does not fully react with the additional superoxide produced under hypoxic conditions. A leakage of superoxide from the erythrocyte is thus detected, resulting in a potential source for oxyradical damage to tissues. Detailed studies on intact erythrocytes as a function of oxygen pressure have now been performed. These studies further delineate the hypoxic stress on erythrocytes and the mechanism for the leakage of superoxide. By centrifugation of samples under various oxygen pressures it was possible to show an enhanced rate of lysis at reduced oxygen pressures with a maximum rate in the region of 25 mm Hg. At much lower pressures where the hemoglobin is mostly deoxygenated the rate of lysis was dramatically decreased with almost no lysis detected even after three days. Lysis is shown to be associated with superoxide membrane damage. The formation of superoxide which does not react with endogenous SOD reaches a maximum value at much lower pressures where most of the hemoglobin is deoxygenated. It is suggested that the leakage at low pressure is associated with the formation of superoxide by oxidation of hemoglobin associated with the membrane.

Cell Hypoxia↗
PubMed1990

Multiple heme pocket subconformations of methemoglobin associated with distal histidine interactions.

Electron paramagnetic resonance spectra of methemoglobin reveal that, in addition to the major tetragonal high-spin aqueous complex and the low-spin hydroxide complex, three other complexes associated with the interaction of the distal histidine are resolved. These are a rhombic high-spin and two classes of low-spin bis-histidine complexes. By freeze-quenching experiments it is shown that the rhombic high-spin and one of the low-spin bis-histidine complexes (B) are at equilibrium with the dominant species. Incubation in the 210-260 K temperature range shifts the total equilibrium toward a low-energy state with the distal histidine coordinated to the iron (complex C).

Animals↗
PubMed1990

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites.

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.

Adenosine Triphosphate↗
PubMed1990

Spin label probes of the environment of cysteine beta-93 in hemoglobin.

The environment of cysteine beta-93 is altered during the oxygenation of hemoglobin. Electron spin resonance was used to probe the hemoglobin conformation in this crucial region on the proximal side of the heme. Spin-labeled hemoglobins in both the R-liganded state [methemoglobin and oxyhemoglobin] and the T-unliganded state [deoxyhemoglobin as well as Ni(II) and Cu(II) substituted hemoglobins] were investigated. Included in this study are iodoacetamide and maleimide labels with different constraints at the point of reaction with the SH-group, as well as a series of pyrrolidinyloxyl maleimide labels of different chain length. From differences in the correlation time of the spin labels it was possible to identify two distinct strongly immobilized configurations in addition to the relatively mobile configuration with the label on the surface of the protein. By dipolar interactions between the spin labels and paramagnetic Cu(II) at the heme center, the relative position of the three orientations for the spin label are defined. Differences are observed between the two hemoglobin conformations with respect to the relative population of the various orientations and with respect to the potential barrier associated with the reorientation of the spin labels.

Animals↗
PubMed1989

Interaction of copper(II) with hemoglobins in the unliganded conformation.

The interaction of exogenous Cu(II) with stable T-state Ni(II)- and Cu(II)-reconstituted hemoglobins has been studied. The relative binding affinities for the two human hemoglobin Cu(II) binding sites are found to be reversed in these hemoglobins relative to native iron(II) hemoglobin A. Nickel hemoglobin, modified by N-ethylmaleimide (NEM), iodoacetamide, and carboxypeptidase A, is used to establish that the observed differences can be attributed to the protein quaternary conformation and not to the metal substitution. Magnetic interactions between the Cu(II) responsible for oxidation and the metal-heme center suggest that the Cu(II) is closer to the heme in T-state hemoglobin than R-state hemoglobin. This finding suggests a pathway for T-state heme oxidation which does not require the beta-93 sulfhydryl group, consistent with rapid Cu(II) oxidation for NEM-reacted deoxyhemoglobin.

Animals↗
PubMed1989

Mössbauer, EPR and NMR studies of the acid-induced reduction and changes in spin state of ferric bleomycin.

Iron-57 Mössbauer, electron paramagnetic resonance (EPR) and H-1 nuclear magnetic resonance (NMR) studies of iron-bleomycin complexes in the pH range from 1.0 to 6.0 are reported. Sequential protonation of the ligands produces a variety of high-spin and low-spin complexes of the metal. Of particular interest is the reversible equilibrium between Fe(III)- and oxygen-stable Fe(II)-bleomycin. Below pH 3.5 Fe(II) complexes form, with maximal reduction occurring at approximately pH 2. At still lower pH, Fe(III) complexes unassociated with bleomycin become dominant. The observed reduction in the absence of exogenous reducing agents suggests the possible involvement of intramolecular autoreduction in bleomycin-mediated DNA degradation.

Bleomycin↗
PubMed1988

Hemoglobin.

Explore the source record for details and available documents.

Animals↗
PubMed1986

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode.

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1985

Low-temperature formation of a distal histidine complex in hemoglobin: a probe for heme pocket flexibility.

Pocket dynamics of horse deoxyhemoglobin and methemoglobin in the temperature range from 80 to 260 K is investigated. In both hemoglobins reversible conversion to a low-spin iron complex is observed at temperatures as low as 210 K. Electron spin resonance (ESR) and Mössbauer data assigned this low-spin iron complex to the coordination of N tau-His-E7 as a sixth nitrogenous ligand. The bonding of this ligand located 4 A from the iron indicates the presence of a thermally available conformation that exhibits a high degree of flexibility in the heme pocket. In deoxyhemoglobin, the formation of the bis(histidine) complex was accompanied by excitations of conformational fluctuations manifested through the temperature dependence of the Mössbauer-Lamb factor. The rate for the formation of this complex, with an associated energy barrier (greater than 60 KJ mol-1), is shown to serve as an index of heme pocket flexibility. Measurements performed on partially liganded (carbonmonoxy) hemoglobin indicate that partial ligation enhances conversion of the unliganded subunits to the bis(histidine) complex, suggesting that pocket dynamics is affected by subunit interactions.

Animals↗
PubMed1985

Temperature dependent spectral changes of iron and nickel hemoglobins and their derivatives.

Temperature dependent absolute and difference spectra for deoxy and oxy human hemoglobin, alpha and beta subunits, NiHbA, carboxypeptidase A treated deoxy HbA and NiHbA have been investigated. It is shown for the first time that the alpha subunits are mainly responsible for the temperature dependent spectral changes in the absorption spectra of Hb in the range from 0 degrees C to 40 degrees C. It has also been found that in the R state the spectral alterations caused by temperature variation are about 85% of those found for the T state of Hb. The value of following the temperature dependence of the porphyrin bands of hemoproteins, as a sensitive probe for subtle changes in the region of the heme, is demonstrated.

Hemoglobins↗
PubMed1985

Interaction between bound cupric ion and spin-labeled cysteine beta-93 in human and horse hemoglobins.

The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the beta-93 cysteine residue. Dipole-dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10-13 A from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 A was determined with human hemoglobin. This distance is consistent with the previously postulated location of the "high affinity" human hemoglobin site near the amino terminus of the beta-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 A was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g11 and A11 for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one beta-chain and the carboxyl terminus of the other beta-chain can explain this shorter distance. This cross-link could involve histidine beta-143, which is one of the ligands thought to be also involved in site 1. Binding to the "high-affinity" site in solution thus stabilizes the "low-affinity" site 2 relative to site 1 explaining the reported interaction between the "high-affinity" and "low-affinity" sites.

Animals↗
PubMed1985

Effects of aging on the lipid order and composition of rat adipocyte ghosts.

An analysis of the cholesterol/phospholipid ratio of adipocyte ghosts from rat epididymal fat pads shows a significant increase with age (P less than 0.005). An attempt to correlate these changes with the order of the lipid matrix was made using the stearic acid spin label 2-(3-carboxypropyl)-4, 4-dimethyl-2-tridecyl-3-oxazolidinyloxyl [I(12,3)]. Although order was negatively correlated with temperature in preparations from both 6- and 24-month-old rats, no effect of age could be detected.

Adipose Tissue↗
PubMed1984

Dynamics of hemoglobin investigated by Mössbauer spectroscopy.

Mössbauer Spectra of Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-Mössbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-Mössbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solution above 200 K where the Lamb-Mössbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.

Animals↗