L. Charles Dickinson

Phospholipid identification and quantification of membrane vesicle subfractions by 31P-1H two-dimensional nuclear magnetic resonance.

An approach to the direct quantification of phospholipids from two-dimensional 31P−1H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.

Cobalt-cytochrome c. II. Magnetic resonance spectra and conformational transitions

Between pH approximately 4 and 10 cobaltocytochrome c (Cocyt-c) gives an electron paramagnetic resonance (EPR) spectrum with g parallel = 2.035, g the perpendicular = 2.223, CoA PARALLEL = 61.4 G, CoA the perpendicular = 49.8 G, NA parallel = 15.3 G, and NA THE PERPENDICULAR = 12.5 G. Comparisons with the EPR spectra of deoxycobaltomyoglobin, deoxycobaltohemoglobin, and model compounds and together with other evidence showed cobaltocytochrome c to have Met-80 and His-18 as its axial ligands. The protons of these ligands are seen as resonances shifted by the ring-current field of the porphyrin in the 300-MHZ 1H nuclear magnetic resonance (NMR) spectra of cobalticytochrome c (Cocyt-c+). The methyl and gamma-methylene protons of Met-80 in this molecule occupy positions with respect to heme c which are somewhat different from those in ferrocytochrome c. The 1H NMR spectra also showed that the methyl groups of Leu-32, Ile-75, Thr-63, thioether bridges, and the porphyrin ring in the cobalt protein are in the same state as in native enzyme; the same is also true for Tyr-59, His-26, and His-33 and also possibly Tyr-67, Tyr-74, and Phe-82. Above pH 11, Cocyt-c is converted to a five-coordinated form having g parallel = 2.026, g the perpendicular = 2.325, CoA parallel = 80 G, CoA the perpendicular approximately 10 G, NA parallel = 17.5 G, and NA the perpendicular not resolved. Below pH 1.0 the EPR spectrum of Cocyt-c is also five-coordinated with g parallel = 2.014, g the perpendicular = 2.359, CoA parallel = 93.8 G, and CoA the perpendicular = 38.8 G. The axial ligands in the alkaline and the acidic forms of Cocyt-c are His-18 and Met-80, respectively. New prominent proton resonance peaks are observed in cobalt-cytochrome c which are either absent or weak in native cytochrome c. These are situated at 3.0, 1.7, and 1.44 ppm, attributable, respectively, to the epsilon-CH2, DELTA-CH2 + beta-CH2, and gamma-CH2 of lysyl residues in random-coil-peptides. From the areas of these peaks, it is estimated that one-two lysyl residues in Cocyt-c have been modified; four-five lysyl residues in Cocyt-c+ have been modified. These alterations of surface charged groups are probably responsible for the lowered reactivity of Cocyt-c with cytochrome oxidase and the lack of reactivity of Cocyt-c+ with several cytochrome reductase systems.

Structure of human deoxy cobalt haemoglobin.

Abstract The structure of human adult deoxy cobalt haemoglobin has been compared to that of the native ferrous form by refinements based on X-ray data to 2.5 A resolution. The two structures were refined in parallel by conventional methods and selected structural differences were measured by a novel difference refinement method applicable to closely related structures. The distance between the metal and the haem plane is 0.33 ± 0.08 A in the cobalt derivative and 0.56 ± 0.03 A in the native. The Co2+HisF8Nϵ bond length is about 0.1 to 0.2 A longer than the Fe2+HisF8Nϵ bond length; the distance of Nϵ from the mean haem plane remains the same in the two structures and the substitution of cobalt for iron produces no significant change in globin structure. The free energy of co-operativity of cobalt haemoglobin is known to be about one-half of that of the native haemoglobin; the reason for this reduction is not evident from the structure of cobalt deoxyhaemoglobin.

Electron paramagnetic resonance of single crystal deoxycobaltohemoglobin

Abstract Single crystals of horse Co Hb were obtained by reduction of Co Hb + crystals with dithionite. Epr measurements showed that the g and Co A tensors are both axial and share the same principal axis systems. Of the four subunits, the “heme” normals of C and d subunits abplane 29 ± 1° from b; they have the same orientation as the hemes in methemoglobin. The normals of “hemes” A and B are 47 above the ab plane as compared to 16° in methemoglobin.

Ionic motions in network polymers containing lithium perchlorate

Abstract Amorphous network polymer electrolytes were derived from poly(propylene oxide) and tris(4-isocyanatophenyl)thiophosphate complexed with lithium perchlorate. The complex impedance diagrams of the higher crosslink density samples were found to be a superposition of two semicircles having different relaxation times. On the other hand, the diagrams of the lowest crosslink density samples were a single semicircle. The Cole-Cole equation was employed to evaluate the relaxation parameters. The higher frequency process was assigned to ionic motions associated with segmental motions of the propylene oxide chain, and the lower frequency process was assigned to ionic motions associated with segmental motions of the urethane group. Dynamic mechanical thermal spectra show two relaxation peaks in the higher cross-link density samples and T1ϱ as a function of temperature obtained by 7Li n.m.r. study has a well defined minimum. These results indicate the correlation between the ionic mobility and the molecular motions of the network system.

Nuclear magnetic resonance studies of the phenylalanine residues of eukaryotic cytochrome c

The resonances of Phe 82 and Phe 10 in the nuclear magnetic resonance spectra of horse cytochrome c are reassigned using nuclear Overhauser enhancements. The reassignments provide new information about the oxidation state linked conformation change of cytochrome c. The region of the protein now known to be affected by the change extends to the part of the protein close to Phe 10.

Mechanism of synergistic stabilization by hindered amines

Abstract Tetramethyl piperidine (TMP) and several derivatives have been studied for their activities in various modes of polymer stabilization. The derivatives are tetramethyl piperidinoxyl (TMPO • ), N -hydroxy tetramethyl piperidine (TMPOH), N -butoxy tetramethyl piperidine (TMPOR) where R is n -butyl and t -butyl, and N -methyl tetramethyl piperidine (TMPMe moiety of Tinuvin 292) as well as some commercial HALS (hindered amine light stabilizer). Inhibition of AIBN initiated cumene oxidation was used to determine free radical scavenging activity; decomposition of t -BuOOH was monitored by iodimetry, gas chromatography, and high pressure liquid chromatography; the rate constant for quenching of carbonyl excited states was measured by inhibition of diethyl ketone initiated photo-oxidation of propylene tetramer; singlet oxygen quenching was determined from the fluorescence intensity of rubrene; the effect on photo-oxidation initiated by photolysis of di- t -butyl peroxide was also studied. TMPOH and TMPMe each scavenges about one peroxyl radical, a combination of the two is strongly synergistic in this action. Further inclusion of TMPO is beneficial. The activity of hydroxyperoxide decomposition decreases in the order TMPOH > TMP > TMPO • > TMPMe. Evidence for Cope reaction between TMPMe and ROOH was seen by ESR. The combination of TMP/TMPO • shows high efficiency in the quenching of diethyl ketone excited states. Other synergistic systems for this process are TMPO • /TMPMe and TMPO • /TMPOR. TMPOH quenches singlet oxygen with an efficacy comparable to that of nickel chelates; it also interferes with di- t -butyl peroxide initiated photo-oxidation. TMPOR shows little or no activity in any of the above tests though it has been proposed to be a key ingredient in a well-known catalytic stabilization mechanism. The results showed that all the TMP derivatives, with perhaps the lone exception of TMPOR, exhibit individually only modest stabilization of some kind. For certain stabilizer functions there are combinations of compounds which are strongly synergistic. If these derivatives were produced during processing or aging of polyolefins containing HALS, their combined actions could explain the outstanding effectiveness of HALS.

Electron paramagnetic resonance of single crystal 15N-nitrosyl-57Fe-myoglobin

Abstract Apomyoglobin and 57Fe-enriched (86%) hemin have been reconstituted and the product crystallized. Subsequent reaction with 15NO (98% enriched) gave 57Mb15NO. Epr of the monoclinic crystals shows a 5.2 ± 0.3 splitting within ± 20° of the a ∗ -axis which is attributable to 57Fe ( I = 1 2 ) splitting. The result suggests a 45% spin density at the iron nucleus in MbNO.

Cobalt cytochrome C: Preparation and characterization☆

Abstract Cobalt cytochrome c has been prepared from porphyrin cytochrome c in water/acetic acid solvent. The dominant band in the electrophoresis of the product at pH7 has the same mobility as the native protein. Dithionite changes the ultraviolet/visible spectrum markedly and generates an epr signal with cobalt hyperfine and other superhyperfine features. Nitric oxide removes part of the epr signal. Fractionation on Amberlite CG-50 under NaCl gradient at pH8.0 yields two major components distinguished by their rates of reactivity to dithionite and electrophoretic mobility. Cobalt cytochrome c is reduced by DPNH cytochrome c reductase to produce the same electron paramagnetic resonance signal as that generated by dithionite.

Circular dichroism and spin-label studies of carp hemoglobin

Abstract Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.