Biomedical subjects

Z Gryczynski

Publications and source records attributed to Z Gryczynski.

46 records · Page 3Linked to original sources

PubMed1993

Effect of intramolecular cross-links on the enthalpy and quaternary structure of the intermediates of oxygenation of human hemoglobin.

We have reported [Bucci, E., Fronticelli, C., & Grycznski, Z. (1991) Biochemistry 30, 3195-3199] that in human and bovine hemoglobins the release of heat at the subsequent steps of oxygenation is not constant. This is especially evident in the binding of the third O2 molecule, which is an endothermic event. This phenomenon was attributed to peculiar conformations of the intermediates of oxygenation, not included in the fundamental R/T transition of the system. To test this hypothesis, we have explored the effect of conformational constraints on the thermodynamics of the intermediates of oxygenation. The assumption was that intramolecular constraints would stabilize the intermediates into conformations similar to the R and T forms reducing the variability of their enthalpies. We have analyzed the temperature dependence of the oxygen binding isotherms of human hemoglobin cross-linked either between the beta 82 or between the alpha 99 lysines by bis(3,5-dibromosalicyl)fumarate. The measurements were perfomred at pH 9.0 in 0.1 M borate buffer in order to avoid thermal effects due to oxygen-linked binding of anions and protons. The data were analyzed singularly by local procedures and simultaneously using global procedures. The two cross-links had opposite effects. The cross-link between the beta-subunits decreased while that between the alpha-subunits increased the endothermic behavior of the third step of oxygenation. Also, the cross-link between the beta-subunits increased the fractional amount of the triligated species at intermediate stages of oxygenation, while that between the alpha-subunits decreased this quantity to hardly detectable values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Recombinant human hemoglobin: modification of the polarity of the beta-heme pocket by a valine67(E11)-->threonine mutation.

Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

A new front-face optical cell for measuring weak fluorescent emissions with time resolution in the picosecond time scale.

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Chemistry Techniques, Analytical↗

PubMed1993

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Binding Sites↗

PubMed1993

Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin.

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems.

Animals↗

PubMed1993

Linear dichroism study of metalloporphyrin transition moments in view of radiationless interactions with tryptophan in hemoproteins.

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe(3+)-PPIX (ferric-heme) and Fe2+CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300-380 nm) and Soret (380-450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300-380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300-380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J. 63, 648-653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 degrees from the alpha-gamma meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.

Binding Sites↗

PubMed1992

Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear.

Energy Transfer↗

PubMed1991

Discontinuous release of heat at successive steps of oxygenation in human and bovine hemoglobin at pH 9.0.

We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.

Animals↗

PubMed1990

Bovine hemoglobin pseudo-crosslinked with mono(3,5-dibromosalicyl)-fumarate.

Under oxygenated conditions bovine hemoglobin reacts with mono(3,5-dibromosalicyl)-fumarate which specifically acylates the EF5 lysines in the beta-cleft of the protein. The chemical modification introduces in the molecule a pseudo-crosslink which hinders the dissociation of the hemoglobin molecule into dimers. Retention time in circulation of the chemically modified bovine hemoglobin, measured in the rat, is increased fivefold with respect to untreated bovine hemoglobin. The oxygen affinity at 37 degrees C and at pH 7.4, has a P50 = 5.4 kPa and a value of n = 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to anions and polyanions whereas it is sensitive to CO2. The Bohr effect is shifted toward the alkaline pH range by 0.5-1: the maximum number of protons released is 1.5/tetramer, similar to normal bovine hemoglobin (1.8 protons/tetramer). Analysis of the binding isotherms, using the two-state Monod-Wyman-Changeux model and fixing the value of the allosteric constant L = 10(5), shows that the oxygen affinity of the T structure is not modified, and that the low oxygen affinity of the system is due to a decrease of the oxygen affinity of the R structure. Analysis using the sequential Adair model shows a modification of the overall binding constants and suggests a redistribution of the intermediate species of oxygenation.

Animals↗

PubMed1990

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin.

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.

Animals↗