Thirty-eighth Annual Meeting March 6-10, 1994 New Orleans Convention Center New Orleans, Louisiana. Thursday Symposia and Posters, Part III

1994 Biophysical Journal  
Two aspects of linkage are addressed. The energetic contribution of a hydrogen bond network in X repressor N-terminal domain to binding of OL1 was examined using thermodynamic 'mutant cycles'. The contribution of N-terminal domain dimerization to binding was also assessed. Binding of x repressor N-terminal domain was examined by calorimetric titration with the synthetic OL1 operator. At 28°C the total binding reaction is enthalpy driven, with a slightly unfavorable entropy. The co-crystal
more » ... ure of N-terminal domain and OL1 shows a hydrogen bonding network between residues Q33, Q44, and the phosphate and base of adenine 2 of the operator. Mutation of either Q33 or Q44 to alanine results in a less favorable binding enthalpy. Changes in binding energetics resulting from mutation of both of these residues are not additive, but rather linked, indicating an interaction between these residues The overall reaction includes a dimerization component. At 28°C dimerization is entropy driven. The thermodynamic parameters for the binding component of the reaction were detenrnined by subtracting the values obtained in the dimerization experiments from the results for the total binding reaction. Both the total reaction and dimerization are strongly temperature dependent. This indicates hydrophobic interactions are important in both dimerization and specific protein-DNA binding. Polyethylene glycol-based polymerization of diaspirn cross-linked hemoglobin (DCLHib), an oxygen-carrying red-cell substitute, offers the benefits of reduced renal clearance and increased retenffon in the vascular circulation. Oxygen equilibrium curves for Poly-DCLHb were slightly left shifted from those of DCLHb with a P5o of 20 mmHg at pH 7.4 and 37°C, a diminished coopretivity (Hill coefficient n =1.7) and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide combination), Poly-DCLHb exhibited a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. Nitric oxide binding to the oxidized forms of modified and unmodified hemoglobin (HbAO) is biphasic, due to the differing reactivities of the a and 3 subunits. The binding of NO occurred at faster rates for the modified hemoglobins. Both proteins exhibited similar propensity for autoxidation and oxidative damage by hydrogen peroxide. The functional and redox properties of Poly-DCLHb are qualitatively similar to those of DCLHb. The reduced Bohr effect can be accounted for on the basis of the blockage of the Bohr residues of DCLHb as result of the polymerization process. PA 19104. (Spon. by J. M. Vanderkooi). Conformational stabilities of protoporphyrin-protoheme hybrid hemoglobins (Hb), in which the protoheme (Fe) in either or both the a-or Isubunits were substituted with protoporphyrin IX (P) (i.e., a(P)2P(Fe)2, a(Fe)2P(P)2 and a(P)2J3(P)2), have been investigated by fluorescence, phosphorescence and fluorescence line narrowing (FLN) spectroscopy. The fluorescence and phosphorescence lifetimes for these hybrids decrease in the order: a, (-PHb > a -PHb > P-PHb at 250 C and 77 K which are interpreted in terms of intersubunit energy and electron transfer. The vibrational features and the energy distribution functions for these hybrids were further studied by FLN spectroscopy. The shape of the distribution function for the 0, 0 transition is approximated by Gaussian functions, centered at 17250 cm-1 for p-PHb and 17380 cm-1 for a-PHb. Temperature dependence of the FLN spectra were different with a transition seen at -50 and 70 K, for porphyrin in a and ,B chain respectively. In addition, step-wise broadening of the individual vibrational lines are observed, indicating differences in flexibility and different phonon coupling for some vibrations of the porphyrin in the two subunits. (Supported by NIH PO1 GM 48130). . (Sponsored by C. Martin) 95% of inter-chain crosslinking as measured by SDS electrophoresis. The thermal denaturation temperature (T") of p,a-XLHbA was 20.2-C higher than the native hemoglobin and 4.3eC higher than either singly crosslinked hemoglobin, o99XLHbA and P82XLHbA. The oxygen affinity of 8,a-XLHbA was the same as the a99XLHbA, and significantly lower than HbA and P82XLHbA. It retained oxygen binding cooperativity. The autoxidation rate of #,o-XLHbA was similar to HbA and e82XLHbA and slower than a99XLHbA. The major product of the reaction of oxyhemoglobin with long diaspirin reagent DBSS was crosslinked between two 0 chains. This crosslinked species produced from oxyhemoglobin had a considerably lower oxygen affinity than HbA and p82XLHbA. The P,0 value was not effected by inositol hexaphosphate. The thermal denaturation temperature was higher than HbA, but about 2.1'C lower than o99XLHbA and 882XLHbA. (Supported in part by Research Corporation grant to K. W. Olsen.) A361 HM PROTEINS II A362 by A. Shrake) Hemoglobin (Hb) interaction with endothelium-derived nitric oxide (NO) is likely responsible for the hemodynamic changes induced by cell-free Hb in vivo. Exposure of nitrosylHb to oxygen or oxyHb to NO may induce Hb oxidation with consequent peroxidative damage to the protein and surrounding tissue. The nitrosyl derivatives of unmodified Hb (HbA0), diaspirin cross-linked Hb (aaHb), and polyethylene glycol polymerized aaHb (PolyaaHb) were each exposed to oxygen in 0.05 M phosphate buffer at pH 7.2. Oxidations were monitored at 27°C in a wavelength scanning spectrophotometer (450-700 nm). Analysis of the spectral data by singular value decomposition (SVD) showed only two components: nitrosylHb and metHb. Muiticomponent analysis showed the oxidations to be biphasic, possibly due to differences in the a and 3 chains, with faster rates for the modified Hb's. Nitroprusside (NTP) oxidations of oxyHb were carried out under similar conditions. Again, only two components were detected, oxyHb and cyanometHb, and the reactions were biphasic and faster for the modified Hb's. NTP oxidation did not produce detectable nitrosylHb. Peroxidative Hb intermediates were not detected in any of the experiments. NO-mediated oxidation of the ferrous heme moiety in Hb may be a normal process that can take place without inducing peroxidative damage. Kg PROTEINS II A383
doi:10.1016/s0006-3495(94)80829-x fatcat:t52b574qs5cmlo5472bnojqfwu