Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.
There have only been a few reports relating to the effect of inhalational anesthetics on the tumor cell morphology in cancer patients undergoing surgery. We hypothesized that some anesthetic agents might influence the spread of unresectable cancer cells and might additionally worsen the condition of the patient due to depressed host immune surveillance. We therefore evaluated the influence of halothane on tumor cell adhesion, which is closely linked to tumor cell metastasis. Human melanoma cells from SK-MEL-37 cell-line were exposed to 4% halothane for 3, 6, 12 or 24 hours, respectively. Furthermore, after 24 hours halothane exposure, they were incubated in a 5% CO2 atmosphere for 12 or 24 hours. The cells were then analyzed using a fluorescence flowcytometer and intercellular adhesion molecule-1 (ICAM-1) expression in SK-MEL-37 cells was quantified as the intensity of fluorescence of ICAM-1 expressed in 10,000 cells. ICAM-1 expression in cells exposed to halothane for 3, 6, 12 or 24 hours was lower than that of non-exposed cells and returned to control level after further incubation in 5% CO2 atmosphere for either 12 or 24 hours. We conclude that halothane might affect the progression of tumor cell metastasis in vitro.
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