Optical rotatory dispersion and circular dichroism of cytochrome oxidase.
Optical rotatory dispersion of the oxidized and the reduced cytochrome oxidase has been measured from 196 to 550 nm. The experimental data have been analyzed in order to separate the extrinsic cotton effects from the rotations of the protein backbone. From the resolution, two cotton effects of similar sign are obtained in the Soret region. For the reduced enzyme, the cotton effect centered at 446.5 nm shows a molar amplitude of 192 k-degrees with a band width of 5.3 nm and rotational strength of 0.25 Debye magneton; the second cotton effect centered at 444 nm shows a molar amplitude of 45 k-degrees with a band width of 12.9 nm and rotational strength of 0.14 Debye magneton. Upon oxidation, the centers of these cotton effects shift to 432 and 423.7 nm. The amplitudes become 114 and 122 k-degrees with band widths of 6.1 and 11.2 nm and rotational strengths of 0.18 and 0.35 Debye magneton, respectively. In addition, a less defined third cotton effect centered at 376 nm appears with an amplitude of 28 k-degrees, band width of 18 nm, and rotational strength of – 0.15 Debye magneton. The circular dichroic curves constructed from the optical rotatory dispersion data by the Kronig-Kramers transform are in general agreement with the experimentally determined spectra. 
 
 
 
The backbone rotations are analyzed and expressed in the terms of Shechter-Blout's parameters. The effective helicity thus computed is 50% for the reduced and 44.5% for the oxidized form. 
 
 
 
The sources of the Soret cotton effects are discussed along with the possibilities of the components present (cytochromes a and a3 and copper), the intrinsic property of the Soret transition, possible splitting of the Soret band, interactions with the protein helix, and heme-heme and heme-copper couplings. The fact that the optical rotatory dispersion and circular dichroism behavior is dependent upon the oxidation state of cytochrome oxidase could arise either from an altered conformation of the protein backbone and/or from an altered interaction with the heme group. Regardless of the possibilities, the geometrical conformation of cytochrome oxidase and the electronic relation between the prosthetic group and the apoenzyme is dependent upon the oxidation state of the enzyme.
Studies on cytochrome oxidase. 3. Ligand reactions and Schiff base formation of heme a.
Abstract Hematin a isolated from purified cytochrome oxidase reacts with poly-l-lysine and with proteins of high lysine content, such as histone, albumin, and globin, in neutral and alkaline media. At pH 11.6, spectral characteristics of these synthetic complexes are strikingly similar to those of cytochrome oxidase. Although absorbance indices differ, the positions of the band maxima are virtually the same in the oxidized, the reduced, the carbon monoxide-reduced, the borohydride-treated, and the borohydride-treated followed by carbon monoxide reduction forms. After borohydride reaction, the heme a in these complexes as well as in cytochrome oxidase is no longer extractable by acid-acetone. On the other hand, these characteristics are not shared when hematin a is replaced by protohematin. Hematin a does not react with histidine, lysine, glutamic acid, aspartic acid, proline, alanine, polyglutamic acid, polyproline, or protamine in either neutral or alkaline media, or with polytyrosine in alkaline solutions under conditions similar to those used for polylysine, i.e. at less than 2 mg of nitrogenous compound per ml. However, the spectra of heme a in the presence of 5% lysine (but not other free amino acids tested, including methionine, histidine, and tyrosine) closely resemble those of the heme a-polylysine complex. Although hematin a reacts with polysarcosine and polyhistidine, the reaction differs from that with polylysine. From the data presented here, together with the behavior of cytochrome oxidase described previously (30), it is tentatively concluded that at least 1 lysine residue of cytochrome oxidase coordinates with the heme iron at pH 11.6, and another lysine residue forms a Schiff base with the formyl group on the porphyrin nucleus. The justification for the conclusion, along with certain reservations, has been pointed out.
Evaluation of Analgesic Effect of Caudal Epidural Tramadol, Tramadol-Lidocaine, and Lidocaine in Water Buffalo Calves (Bubalus bubalis)
Aim of this study was to compare the analgesic effect of tramadol and a combination of tramadol-lidocaine with that produced by lidocaine administration in the epidural space in buffalo calves. In a prospective randomized crossover study, ten male buffalo calves were used to compare the epidural analgesic effect of tramadol (1 mg/kg) and tramadol-lidocaine combination (0.5 mg/kg and 0.11 mg/kg, resp.) with that produced by 2% lidocaine (0.22 mg/kg). Loss of sensation was examined by pin-prick test. Onset time, duration, and degree of analgesia and ataxia were recorded after each treatment. Heart rate (HR), respiratory rate (RR), rectal temperature, and haematobiochemical parameters were recorded after all treatments. Time to onset and duration of analgesia, respectively, were as follows: tramadol 11 ± 2 min and 208 ± 15 min; tramadol-lidocaine 6 ± 2 min and 168 ± 9 min; lidocaine 4 ± 1 min and 67 ± 13 min. Onset time and duration were significantly longer with tramadol than the other treatments. Duration was significantly longer with tramadol-lidocaine than lidocaine. Ataxia was mildly observed in tramadol-lidocaine and was moderate in lidocaine. HR, RR, and rectal temperature did not differ significantly from baseline after any treatment. Haematobiochemical parameters returned to basal levels by 24 h after all treatments. This combination might be clinically useful to provide analgesia in buffalo for long-duration surgical procedures.