Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

Temperature dependent UV-Vis spectral changes in hydrogen-and deuterium-bonded photosynthetic reaction centers of Rhodobacter sphaeroides

The UV-Vis absorption spectra of detergent-isolated hydrogen-and deuterium-bonded reaction centers (RCs) from Rhodobacter sphaeroides PUC 705Ba were examined as a function of temperature between 20 and 55 °C. The enthalpy and entropy of denaturation for the specimens was determined, revealing that their process of thermal denaturation is significantly different. Deuterium-bonded RCs are most stable at 37 °C, rather than at room temperature, and undergo a “cold denaturation” as the temperature is lowered to room temperature. At room temperature the addition of 1,3,5-heptanetriol brought the deuterium-bonded RC back to its more stable configuration. Hence the hydrogen bonding interactions in the RC do influence its conformation and this is reflected in the microenvironment of its associated pigments.     

DNA assisted fragmentation of nickel nanoparticle clusters and their spectral properties

Nickel nanoparticle (NiNP) clusters in the range of 60-70 nm size on interaction with herring-sperm DNA (B-DNA) form a self-assembled duplex helix DNA structure with fragmented NiNPs as small as 5-15 nm, as evident from atomic force microscopic studies. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images also corroborate the findings. The properties of these self-assembled NiNPs-DNA structures have been further investigated by UV-visible, emission and circular dichroic (CD) spectral studies.     

Temperature dependent membrane potential changes in snail neurons and their relation to active ion transport.

The mechanisms underlying the temperature response of the resting membrane potential (RMP) were investigated in 3 identified neurons of the buccal ganglion of Helix pomatia. Lowering the temperature evoked a decrease of the RMP and an increase in membrane resistance, and vice versa. The temperature response of the RMP had an equilibrium potential of ca, -60 mV. It is essentially evoked by changes in the potassium conductance. Indications of an electrogenic sodium transport were not detected.     

Temperature modulation of bovine hemoglobins

The functional properties of hemoglobin from Egyptian water buffalo have been characterized as a function of pH, temperature and chloride concentration. Alongside overall similarities shared with ox and Arctic ruminant hemoglobins, hemoglobin from buffalo shows significant differences with respect to the effect of temperature. The results obtained may suggest that the limited effect of temperature on oxygen binding recently reported for ox hemoglobin could be regarded as an interesting case of a reminiscence of a past glacial age.     

pH dependent conformational changes within the iron responsive element

The expression of several mRNAs related to iron importation, storage and utilization within mammalian cells are regulated through interactions of iron regulatory proteins with an iron responsive element, an RNA hairpin with a bulged C. A dimethylsulfate modification interference assay was used to demonstrate that the iron responsive element undergoes significant pH dependent conformational changes. Specifically, it was demonstrated that the phylogenetically conserved A within the hairpin loop and an intra-loop C-G base pair are highly sensitive to changes in pH. The conserved C of the bulged loop does not significantly affect the pH dependent conformational changes of the hairpin loop. These studies have structural implications for an RNA-protein interaction that is critical to mammalian iron regulation.     

Temperature and pH dependent changes of immunoglobulin G structure.

1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.     

Temperature dependent structural changes of intraphage T7 DNA

The phenomena connected with the first phase transition step of the native T7 phage at 40C–65 C have been studied using various methods. In this temperature range a) the optical melting curve shows an absorption decrease, b) the maximum of the small-angle X-ray scattering characteristic for DNA packing disappears, c) there is a drop of biological activity and d) there are changes in the structure of the difference absorption spectra of native phages versus isolated DNA. All data are interpreted assuming a structural change of the DNA due to the release of its protein coat towards the end of the first phase transition step (at 60–65 C in the case of M9 buffer). Above this temperature the intraphage DNA packing appears to be destroyed and the DNA structure seems to be similar to that in DNA solution.     

Temperature dependent hysteresis-like changes of motility parameters in human spermatozoa

We studied the impact of temperature changes on selected parameters of normozoospermic spermatozoa motility. The examinations were carried out in the temperature range of 11 degrees C to 21 degrees C both during cooling and heating. We found that a phenomenon of hysteresis, i.e. alternate means of changes in velocity straight linear and lateral head displacement was obtained both at cooling and at heating. This phenomenon was not found for other parameters examined.     

Neutral changes during divergent evolution of hemoglobins

Summary A comparison of the mRNAs for rabbit and human-hemoglobins shows that synonymous changes in codons have accumulated three times as rapidly as nucleotide replacements that produced changes in amino acids. This agrees with predictions based on the so-called neutral theory. In addition, seven codon changes that appear to be single-base changes (according to maximum parsimony) are actually two-base changes. This indicates that the construction of primordial sequences is of limited significance when based on inferences that assume minimum base changes for amino acid replacements.     

Temperature dependent changes of cell growth parameters in Tetrahymena

Cytophotometric studies revealed gross amounts of RNA and protein to be negatively correlated with incubation temperature. The rate of [³H]-uridine incorporation into TCA-insoluble material of synchronously growing cells followed a cyclic pattern during the cell cycle and was correlated to the event of the S phase.     

Temperature dependent permeability changes of Bacillus subtilis and incorporation of nucleotides into DNA

Cells of $\text{B. subtilis}$ exposed to temperatures between 0 and 5 C are permeable to small molecules not normally able to pass through the cell envelope. As a result, deoxyribonucleotide triphosphates (dXTPs) are incorporated into DNA if the reaction mixture contains all four dXTPs. Since this incorporation is insensitive to 6-(p-hydroxyphenylazo)-uracil and is not observed in DNA Polymerase I mutants, we conclude it reflects DNA repair rather than the DNA replication which can be observed in cells permeabilized with toluene.     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.     

Chemically modified porcine hemoglobins and their biological properties

Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model.     

Catalytic strategies of the non-heme iron dependent oxygenases and their roles in plant biology

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Ligand-induced spectral changes in cytochrome c oxidase and their possible significance.

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.