Biomedical subjects

J S Philo

Publications and source records attributed to J S Philo.

46 records · Page 3Linked to original sources

PubMed1993

Densimetric determination of equilibrium binding of sucrose octasulfate with basic fibroblast growth factor.

Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. Sucrose octasulfate (SOS), which has a chemical structure resembling the repeating unit of heparin, has also been shown to enhance stability of basic FGF against thermal denaturation and to induce a small conformational change. We have examined SOS binding to bFGF using equilibrium dialysis. The difference in SOS concentration across the dialysis membrane was measured using a precision density meter, since the density of SOS differs greatly from that of water. With care, this densimetric technique can measure binding with a precision of +/- 0.1 mol/mol using about 2 mg/ml of protein. These results show that the binding saturates at 2 mol of SOS per mole of bFGF as the SOS concentration increases to 3.6 mM or higher. The effect of SOS on the thermal stability of bFGF was examined using denaturation at a constant heating rate, by both turbidity and differential scanning calorimetry. Since the thermal denaturation is irreversible, the temperature where aggregation abruptly increases was taken to indicate the onset of denaturation. This temperature increased by approximately 12 degrees C as the SOS concentration increased from 0.018 to 3.6 mM and remained constant above 3.6 mM, consistent with our binding data if the binding is specific to the native state.

Amino Acid Sequence↗

PubMed1992

Kinetics of oxygen binding and subunit assembly for the hemoglobin alpha subunit.

A thorough kinetic characterization of the O2-binding and self-association reactions of alpha-subunits of human hemoglobin A has been performed. All of the rate constants for a five step reaction model linking the monomer-dimer reaction to the O2-binding steps have been determined for the first time. Our analysis of the ligand binding reaction shows that both monomer and dimer have nearly identical intrinsic O2-association and dissociation rate constants and therefore identical affinities for oxygen. During this investigation we discovered a small absorbance difference between the oxy-monomer and oxy-dimer alpha-subunits. This difference spectrum enabled direct measurements of the alpha O2 self-association reaction. We find an association rate constant of, 2.0 10(5) M(-1)s-1, similar to that for other subunit assembly processes in the hemoglobin system. Our results also suggest that the deoxy-subunit assembly kinetics must be similar to that for the oxy-subunit. These kinetic results together with the equilibrium constants obtained for these solution conditions by Ackers and coworkers provides, for the first time, a complete kinetic and thermodynamic description of all the intrinsic ligand binding and association reactions for alpha-subunits.

Chemical Phenomena↗

PubMed1991

Ca2+ depletion from the photosynthetic water-oxidizing complex reveals photooxidation of a protein residue.

A new intermediate in the water-oxidizing reaction has been observed in spinach photosystem II (PSII) membranes that are depleted of Ca2+ from the site which is conformationally coupled to the manganese cluster comprising the water-oxidizing complex (WOC). It gives rise to a recently identified EPR signal (symmetric line shape with width 163 +/- 5 G, g = 2.004 +/- 0.005), which forms in samples inhibited either by depletion of Ca2+ [Boussac, A., Zimmerman, J.-L., & 28, 8984-8989; Sivaraja, M., Tso, J., & Dismukes, G.C. (1989) Biochemistry 28 9459-9464] or by substitution of Cl- by F- (Baumgarten, Philo, and Dismukes, submitted for publication). Further characterization of this EPR signal has revealed the following: (1) it forms independently of the local structure of the PSII acceptors; (2) it arises from photooxidation of a PSII species that donates an electron to Tyr-Z+ or to the Mn cluster in competition with an exogenous donor (DPC); (3) the Curie temperature dependence of the intensity suggests an isolated doublet ground state, attributable to a spin S = 1/2 radical; (4) the electron spin orientation relaxes 1000-fold more rapidly than typical for a free radical, exhibiting a strong temperature dependence of P1/2 (half-saturation power approximately T3.4) and a broad inhomogeneous line width; (5) it yields an undetectable change in the magnetic susceptibility upon formation by a laser flash; (6) it disappears in parallel with release of Mn during reduction with NH2OH, indicating that it forms only in the presence of the modified Mn cluster. (ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1990

Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue.

New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl- facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl- depletion or F- substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl- and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 +/- 0.005, a symmetric line shape with line width = 16 +/- 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 +/- 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (less than 1 mu B2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 A of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorides↗

PubMed1990

Kinetic investigations of the quaternary enhancement effect and alpha/beta differences in binding the last oxygen to hemoglobin tetramers and dimers.

Analysis of O2 binding equilibria by two independent groups has suggested that the affinity for binding the fourth O2 to Hb tetramers is very high, about 800-1200 cal/mol higher than that of dimers (Chu, A. H., Turner, B. W., and Ackers, G. K. (1984) Biochemistry 23, 604-167; Di Cera, E., Robert, C. H., and Gill, S. J. (1987) Biochemistry 26, 4003-4008). Recently, Gibson and Edelstein challenged the reality of the quaternary enhancement effect, based on kinetic data (Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516-519). However, these studies failed to directly address the key issue of the relative affinities of dimers and alpha 2 beta 2(O2)3. Furthermore, the extent to which alpha/beta differences influence these results remains an open question. Using partial laser photolysis and O2/CO replacement techniques we have, for the first time, resolved the rates of O2 association and dissociation to both alpha and beta chains within "R state" tetramers and dimers. We find that the beta chains are faster than alpha for both O2 binding (approximately 2-fold) and release (approximately 3-fold). The kinetically determined O2 affinities derived from these data are essentially identical for dimers and alpha 2 beta 2(O2)3. That is, the data do not show significant quaternary enhancement and suggest that the equilibrium data have both overestimated the affinity of alpha 2 beta 2(O2)3 and underestimated the affinity of dimers. The significance of and possible origins for the discrepancy between equilibrium and kinetic data are discussed.

Carbon Monoxide↗

PubMed1988

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.

Algorithms↗

PubMed1985

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins.

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.

Calorimetry↗

PubMed1984

Diamagnetism of human apo-, oxy-, and (carbonmonoxy)hemoglobin.

In recent years, a controversy has arisen over the magnetic properties of oxyhemoglobin (HbO2) and (carbonmonoxy)hemoglobin (HbCO). At present, it is unclear which, if any, conditions give a completely diamagnetic state for the heme-ligand complex which can be used as a diamagnetic reference state. In order to establish a diamagnetic reference independent of assignments of electronic configurations, we have measured the magnetic susceptibilities of apohemoglobin solutions and powdered iron-free protoporphyrin IX. We have also reexamined the magnetism of HbO2 and HbCO solutions at 20 degrees C and at several ionic strengths. We find no difference in magnetism between HbO2 and HbCO and no changes in their magnetism with solution conditions. Furthermore, relative to the new (apohemoglobin + porphyrin) diamagnetic reference, our data are consistent with complete diamagnetism for both HbO2 and HbCO under all conditions we have studied. Our data imply that any low-lying triplet state must lie at least 900 cm-1 above the diamagnetic ground states. These results disagree strongly with reports of substantial room temperature paramagnetism for HbO2 and a smaller paramagnetism for HbCO which disappears at high ionic strength [see Cerdonio, M., Morante, S., Vitale, S., Giacometti, G., & Brunori, M. (1982) in Hemoglobin and Oxygen Binding (Ho, C., Ed.) pp 63-68, Elsevier/North-Holland, Amsterdam, and references cited therein].

Apoproteins↗

PubMed1981

Association-dependent absorption spectra of oxyhemoglobin A and its subunits.

A number of factors which lower the oxygen affinity of hemoglobins are also known to produce shifts of the absorption band maxima of the oxyheme. We have studied the variation of the absorption spectra of oxygenated alpha SH and beta SH subunits of human Hb A as a function of their state of association (i.e. alpha, alpha 2, beta, beta 4, alpha beta, or alpha 2 beta 2), and have attempted to correlate the spectral changes with changes to O2 affinity. These studies were carried out under solution conditions (0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, 10 degrees C) where detailed thermodynamic data for subunit association and oxygen binding are available (Ackers, G. K. (1980) Biophys. J. 32, 331-346). Concentration-difference spectra reveal that the visible and Soret absorption band maxima of beta O2 are slightly red shifted relative to beta 4O8. A unique feature of this spectral change is that the red shift is accompanied by an increase in the ratio of the peak absorbances of the visible alpha and beta spectral bands. By measuring the spectral change as a function of concentration, an association constant of 6.4 +/- 1.9 X 10(15) M-3 was determined for the 4(beta O2) in equilibrium beta 4O8 equilibrium. In contrast, no spectral differences were found between alpha O2 and alpha 2O4 or between oxy alpha beta dimers and oxyHb. Mixing experiments show that the spectrum of oxyHb differs from the average of either alpha O2 + beta O2 or alpha O2 + beta 4O8, but is closer to the former. A comparison between these spectral data and the reported O2 affinities of these species shows that affinity and oxyheme spectra are not correlated.

Carboxyhemoglobin↗

PubMed1977

Kinetics of hemoglobin-carbon monoxide reactions measured with a superconducting magnetometer: a new method for fast reactions in solution.

A new technique for measuring fast reactions in solution has been demonstrated. The changes in magnetic susceptibility during the recombination reaction of human hemoglobin with carbon monoxide after flash photolysis have been measured with a new high-sensitivity instrument using cryogenic technology. The rate constants determined at 20 degrees (pH 7.3) are in excellent agreement with those obtained by photometric techniques [Gray, R.D. (1974) J. Biol. Chem. 249, 2879-2885]. A unique capability of this new method is the determination of the magnetic susceptibilities of short-lived reaction intermediates. The magnetic moment of the intermediate species Hb4(CO)3 was found to be 4.9+/-0.1 muB in 0.1 M phosphate buffer by partial photolysis experiments. This value agrees with the predictions of two-state allosteric models of cooperativity in hemoglobin. Possible applications and improvements in this technique are discussed.

Carbon Monoxide↗