Unfolding pathway of myoglobin: molecular properties of intermediate forms

The guanidine-induced unfolding of myoglobin as well as apomyoglobin has been found to involve the occurrence of at least a molecular intermediate observed at low denaturant concentrations, the molecular properties of which resemble those possessed by the acid-denatured form of the protein. The two partially unfolded forms show the same secondary structure and similar tryptophanyl fluorescence emission and polarization but exhibit marked differences in the tyrosine contributions to the near-ultraviolet circular dichroism and in the degree of solvent accessibility to tyrosyl residues. The molecular characterization of the two structural forms indicates that acids disorganize the 80-146 molecular domain identified in the myoglobin molecule to a great extent with respect to that induced by low guanidine concentration, whereas the structure of the 1-79 domain appears to be quite similar in the two molecular forms.     

Effect of gamma-irradiation on some biological and physico-chemical properties of polyene antibiotics]

Deep effect of gamma-rays on polyenic antibiotics was studied. It was shown that gamma-radiation induced radiation-chemical oxidation of the substances. The chromatographic analysis showed that the levorin degradation products were identical to the polyenic products of the antibiotic oxidative destruction. As for mycoheptin and amphotericin B, destruction of their molecules to non-polyenic products was observed. It was found that toxicity of the levorin aromatic heptaen did not practically change after gamma-irradiation in high doses. The toxicity of mycoheptin and amphotericin B, non-aromatic heptaens increased after exposure to high doses of gamma-rays.     

Effects of gamma-irradiation on the electrokinetic properties of purple membranes

The effect of gamma-irradiation (5, 10 and 15 Gy) on the kinetic surface charge of purple membranes (PM) was followed by means of particle microelectrophoresis. The changes in electrophoretic mobility (EPM) were examined at 2, 5 and 26 h, respectively, following irradiation of native PM, and at 2, 26, 50 h and 5 days following irradiation of delipidated PM. It was concluded that the high inhomogeneity of the suspension, even after sonication, largely affects the measured zeta-potential. The 15-Gy treatment significantly increased the net negative surface charge density at 5 and 26 h after irradiation of native PM. However, the opposite effect of approximately twofold reduction of EPM values was derived from simultaneous studies concerning their delipidated form. Low irradiation doses clearly induced an enhancement of negative surface charge density at 2 h post-exposure as well as the formation of unstable structures of delipidated PM. The changes in electrokinetic properties might reflect the specific aggregate formation in both native and delipidated PM. It was suggested that the effect observed of both types of PM was mainly a structural phenomenon possibly related to the modification of functionally active residues. Received: 15 May 1997 / Accepted in revised form: 8 January 1998     

Effect of gamma-irradiation on dye-DNA binding.

The effect of gamma-rays on the binding of proflavine and acridine orange to DNA was investigated by spectrophotometry. The effect of irradiation was observed on the buffered solutions of the free dye and free DNA. A dose of about 35 krad caused a hyperchromicity of 30-40 per cent to the DNA peak at 258 nm, while the same dose introduced a hypochromic effect to the monomer peaks of the dyes by 30 per cent. This implied that gamma-rays have an effect of decreasing the monomer concentration of free-day molecules in solution. From the results, we conclude that more dye is bound to the changed conformation of dye-bound DNA on irradiation. Scratchard-binding isotherms drawn for the unirradiated and irradiated complexes of Pf-DNA showed interesting differences. Similar isotherms could not be obtained for the acridine orange-DNA system.     

Effect of gamma-irradiation on microtubule assembly in vitro

Microtubular protein was exposed to gamma-radiation from 500 to 1000 Gy, Within that dose range its polymerization ability was decreased by 20-60 per cent when samples were irradiated in assembled state, and by 40-75 per cent when irradiated in unassembled state. Microtubules assembled from irradiated subunits were shorter and of more uniform lengths than control microtubules. For the dose of 1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.     

Catalytic properties of testicular hyaluronidase after gamma-irradiation

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.     

Effect of gamma-irradiation on oocysts of Eimeria necatrix.

Effect of gamma radiation on oocysts of Eimeria necatrix was investigated. It was observed that oocysts exposed to 200 kR or above did not sporulate. Irratiation at 10-150 kR caused a progressive decrease in sporulation. Irradiation affected normal development of unsporulated oocysts as the zygote protoplasm divided into unequal masses or was shattered into granules. Increase in the intensity of irradiation of sporulated oocysts resulted in the progressive decrease in severity of the resultant infections in chicks and their effects - mortality, type of lesions developed, total oocyst production and immunity produced - were comparable with infections induced by decreasing the number of unirradiated oocysts. Infection produced by 1000 unirradiated oocysts was comparable with that resulting from 50 000 oocysts irradiated at 25 kR. Infection obtained with 20 000 unexposed oocysts approximated to that produced by 50 000 oocysts irradiated at 2-5 kR. It was concluded that irradiation abolished infectivity of the oocysts/sporozoites rather than bringing about attenuation of the parasite.     

Salt-induced refolding of myoglobin at acidic pH: molecular properties of a partly folded intermediate.

The molecular properties of the salt-induced partly folded acidic state of apomyoglobin as well as myoglobin were investigated by fluorescence and circular dichroism of the extrinsic fluorophore 1,8-anilinonaphthalenesulfonate. The occurrence of a fluctuating tertiary structure ("molten globule") at acidic pH in the presence of salt was suggested by the disappearance of the dichroic activity of the fluorophore bound to the partly folded protein. Moreover, the structure of the intermediate is not influenced by the presence of heme, thus suggesting that heme is not crucial in the early stage of myoglobin folding.     

Effect of peripheral trifluoromethyl groups in artificial iron porphycene cofactor on ligand binding properties of myoglobin

An iron porphycene, a structural isomer of iron porphyrin, with trifluoromethyl groups at the peripheral position of the framework was incorporated into sperm whale apomyoglobin. The prepared myoglobin shows the higher O(2) affinity than the native protein. However, the oxygen affinity of the reconstituted myoglobin is lower than that of the myoglobin having an iron porphycene without trifluoromethyl groups, which is mainly originated from the enhancement of the O(2) dissociation. The CO affinity of the myoglobin with the trifluoromethylated iron porphycene is similar to that observed for the reference protein having the iron porphycene without trifluoromethyl groups, although their C-O stretching frequencies are significantly different. The relationship between the electronic states of the porphycene ring and the ligand bindings is discussed.