Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).

A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins.

The nuclear Overhauser effect (NOE) was used to investigate heme orientation and to obtain assignments for all resolved resonances in the 1H-NMR spectrum of met-cyano Chironomus thummi thummi monomeric hemoglobins III and IV (Hb III and Hb IV). The only non-heme resolved resonance was found to be from Phe-38 (CD1), and NOE dipolar connectivity between this resonance and the heme 5- and 8-methyls was used to establish the absolute orientation of the heme for each heme-insertion isomer present. The assignments of resonances and heme disorder permitted structural comparisons between the various components, including those due to a point mutation in Hb III. Finally, the characteristic differences of NOE patterns to amino-acid protons from substituents on heme pyrroles I and II formed the basis for assigning resonances and heme orientation relative to native Hb IV for deuterohemin-reconstituted Hb IV, for which there are no X-ray data available.

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.

Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy.

The haem-rotational disorder (insertion of haem into globin rotated about the alpha, gamma-meso axis by 180 degrees) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two Fe-C-N bending vibrations at 401 cm-1 and 422 cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One Fe-C-N bending mode at 422 cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t----r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the Fe-N epsilon(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single Fe-C-N bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the Fe-C-N bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the Fe-C-N and vinyl bending modes. The Fe-N epsilon(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe/57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange.

Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins. Dynamic stability of the heme pocket as monitored by labile proton exchange.

The 1H nuclear magnetic resonance spectral characteristics of the cyano-Met form of Chironomus thummi thummi monomeric hemoglobins I, III and IV in 1H2O solvent are reported. A set of four exchangeable hyperfine-shifted resonances is found for each of the two heme-insertion isomers in the hyperfine-shifted region downfield of ten parts per million. An analysis of relaxation, exchange rates and nuclear Overhauser effects leads to assignments for all these resonances to histidine F8 and the side-chains of histidine E7 and arginine FG3. It is evident that in aqueous solution, the side-chain from histidine E7 does not occupy two orientations, as found for the solid state, rather the histidine E7 side-chain adopts a conformation similar to that of sperm whale myoglobin or hemoglobin A, oriented into the heme pocket and in contact with the bound ligand. Evidence is presented to show that the allosteric transition in the Chironomus thummi thummi hemoglobins arises from the "trans effect". An analysis of the exchange with bulk solvent of the assigned histidine E7 labile proton confirms that the group is completely buried within the heme pocket in a manner similar to that found for sperm whale cyano-Met myoglobin, and that the transient exposure to solvent is no more likely than in mammalian myoglobins with the "normal" distal histidine orientation. Finally, a comparison of solvent access to the heme pocket of the three monomeric C. thummi thummi hemoglobins, as measured from proton exchange rates of heme pocket protons, is made and correlated to binding studies with the diffusible small molecules such as O2.

The effect of cadmium ions on 2,3-bisphosphoglycerate in erythrocytes studied with 31P NMR.

The interaction of cadmium ions with human red blood cell (RBC) 2,3-bisphosphoglycerate (DPG) wasa studied by 31P NMR. 31P NMR spectra and 31P T1 and 31P T2 relaxation times give evidence for cadmium-2,3-bisphosphoglycerate complexation in aqueous solution. 31P NMR spectra indicate the occurrence of a similar complexation in RBC cryolysates. The lag phase (constant polyphosphate level) prior to DPG hydrolysis in incubated red blood cells is lengthened in the presence of cadmium.

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin.

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.

Assignment of the Fe-N epsilon (His) stretching mode in the resonance Raman spectra of a monomeric insect cyanomethaemoglobin.

Resonance Raman (RR) spectra of the monomeric cyanomethaemoglobin CTT III from insect larvae of Chironomus thummi thummi are shown for the range of 200-550 cm-1. By iron and cyanide isotope exchange a line varying between 307 cm-1 for 57Fe-13C15N and 311 cm-1 for 54Fe-12C14N, has been assigned to the Fe-N epsilon stretching mode of this haem complex. The substitution of 54Fe for 57Fe has no effect on the Fe-C = N bending mode whereas it affects the Fe-CN stretching mode.

The influence of infusion rate on the acute intravenous toxicity of phytic acid, a calcium-binding agent.

The intravenous toxicity of phytic acid (inositol hexakisphosphate, IHP) has recently become of interest because of the potential for IHP incorporation into red blood cells to achieve a therapeutically useful shift in the hemoglobin-oxygen dissociation curve (Gersonde, K. and Nicolau, C., Blut, 39 (1979) 1). The observed acute intravenous toxicity of IHP in rodents is consistent with its recognized capacity to bind calcium. The toxic manifestations of intravenous IHP are a function of rate of infusion as well as total dose, with some seemingly anomalous variations which may related to compensatory mechanisms. The data suggest that significant alterations of plasma calcium and the toxic potential of such alterations are not likely to result from the administration of red blood cells with IHP incorporated.

A new method of mechanical circulatory support with an implantable multichamber pump system (IMPS): presentation and first experimental results.

This paper presents a new cardiac support device for left ventricular failure which consists of two inflatable bellows positioned dorsally and ventrally to the left ventricle. The implantable multichamber pump system (IMPS) is driven by a pneumatic pump system and controlled by a microcomputer using ECG-trigger and pacemaker modules. It was implanted via thoracotomy in 8 pigs. The circulatory parameters were measured in the animals on beta-blockers, with cardiac failure and in ventricular fibrillation with an activated (IMPS on) and deactivated (IMPS off) system. IMPS significantly increased the left ventricular pressure (LVPsys IMPS off: 63 +/- 6 mmHg vs IMPS on: 96 +/- 8 mmHg) and the blood pressure in the common carotid artery (BPca, IMPS off: 69/38 mmHg vs IMPS on: 95/40 mmHg). The IMPS proved to be highly efficient in the therapy of animals with acute cardiac failure and in ventricular fibrillation in the experimental model. Apart from its efficiency the advantages with this system are the ease of handling and its high biocompatibility due to the lack of contact with circulating blood.

[Error analysis, biological modification factors and variance of peri-ungual video-capillary microscopy]. / Fehleranalyse, biologische Einflussfaktoren und Varianz der periungualen Videokapillarmikroskopie.

Error analysis or the evaluation of the precision of microscopic measurements must distinguish between technical errors inherent in the measuring system, and biological variability. The technical error inherent in the overall system in the case of linear measurements is smaller than the lengths to be measured by 1 or 2 orders of magnitude. Thus, the erythrocyte column lengths can be correctly, reproducibly and linearly quantified over the entire measuring range. The biological influencing factors can largely be taken into account or excluded by suitable standardisation of the measuring process. Despite a considerable individual fluctuation in the erythrocyte velocity, there is, on average, no significant dependence of the measuring parameter within the daily profile or from day to day. Differences in capillary perfusion in the presence of diseases associated with microcirculatory disorders or a therapeutic influence on erythrocyte velocity can thus be reliably demonstrated.

Effect of cadmium ions on dioxygen affinity and polyphosphate activity of human red blood cells.

The effect of cadmium ions on the dioxygen affinity, the time-dependent depletion of intracellular polyphosphates, and the elongation of human red blood cells (RBC's) was examined. The incubation of RBC's in the presence of 1 mM Cd2+ at 37 degrees C for more than one hour results in a decrease of the p50 value by 2.5-3.0 mmHg in comparison to controls. The p50 of stripped (phosphate-free) hemoglobin is not affected by the presence of 1 mM Cd2+ (p50 = 4.8 mmHg at pH 7.2 and 37 degrees C). Experiments with RBC cryolysates demonstrate an apparently competitive effect of 2.3-bisphosphoglycerate (DPG) with cadmium ions on the dioxygen affinity. From 31P NMR spectra, 31P T1 relaxation, and 31P T2 relaxation behavior a more direct evidence for DPG-Cd2+ complexation is obtained. 31P NMR spectra of RBC cryolysates also indicate DPG-Cd2+ complexation. The hydrolysis of free polyphosphates in RBC's incubated at 37 degrees C as monitored by 31P NMR spectra can be noticed after a three-hour lag phase (constant polyphosphate level). This lag phase is lengthened from three hours to four hours in the presence of Cd2+ ions. RBC elongation, as a measure of deformability, decreases slightly upon incubation with 1 mM Cd2+.

Echo shape analysis and image size adjustment on the level of echoes: improvement of parameter-selective proton images.

Parameter-selective imaging for tissue characterization is performed at low magnetic fields (0.24-0.28 T) employing echo trains with a large number of echoes (less than or equal to 50), high-power pulses, and the projection reconstruction method. Preprocessing of the raw echoes is important for the quality of the echo images which are the bases for further evaluation and generation of parameter-selective images. Two new algorithms are implemented in the image preprocessing routine TOMIKON: Echo shape analysis and image size adjustment. Echo shape analysis allows the detection and the removal of spikes in the echo signals to avoid image artifacts. Image size adjustment is performed by expanding and contracting the time basis of the echoes resulting in a size normalization of the echo images, an indispensable condition for generating diffusion images.

Incorporation of inositol into intact red blood cells. II. Enhancement of gas transport in inositol hexaphosphate-loaded red blood cells.

The gas-transport function of red blood cells which have incorporated inositol hexaphosphate is significantly improved by fusion with effector-loaded lipid vesicles. "Right-shifts" of the O2-binding curves of inositol hexaphosphate-loaded red blood cells with half-saturation pressures at 37 degrees C up to 98 mmHg are observed. The transformation of 56% of the intracellular haemoglobin into the low affinity state corresponds to an optimum increase of the O2-release capacity of 270%. The CO2 transport is also correspondingly enhanced. The treated red blood cells show no inositol hexaphosphate depletion during a reasonable time.