PubMed · 7430380
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S M Lewis, N K Shinton. 1980. Cyanmethaemoglobin reference preparations.. https://doi.org/10.1136/jcp.33.7.700-c
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An immunohistochemical investigation of postmortem lividity was performed to illuminate localization of hemoglobin (Hb) and the mechanism of fixed lividity. The fixed lividity was defined as an unfading phenomenon by thumb finger pressure. Skin specimens were taken from 68 autopsy cases 7.5-336 h (2 weeks) postmortem. Localization of Hb of the specimens was examined by a labeled streptabidin biotin (LSAB) method using polyclonal (rabbit antihuman hemoglobin antibody) and monoclonal (mouse anti-human hemoglobin monoclonal antibody) antibodies. Positive staining for Hb was observed in various sites of the skin, i.e. in only intravascular erythrocytes, in vascular walls and perivascular tissue including sweat glands and sebaceous glands, in the dermal connective tissue, and in almost all of skin tissue except the horny layer. The diffusion of Hb into skin tissue was observed in 20 of 41 displaceable lividity cases (49%) and 11 of 27 fixed lividity cases (41%). Compared to displaceable lividity, superficial plexi in fixed lividity were filled with erythrocytes, which were markedly immunodetected. These findings support the hypothesis that the fixation of lividity is not due to diffusion of Hb into skin tissue but hemoconcentration in blood vessels.
Ultraviolet resonance Raman (UVRR) spectra have been obtained for cyanometHb (Hb = hemoglobin) hybrid tetramers representing several ligation microstates, using dimer interchange techniques and spectral subtraction. Relative to fully ligated tetramers, Hb(CN)4, the monoligated hybrids and deoxyHb all show fully developed T/R difference UVRR bands which are associated with T state quaternary contacts across the alpha 1 beta 2 interface, involving the Trp beta 37 and Tyr alpha 42 residues. Triligated species show quite different signals, arising from the interior residues Trp alpha 14 and/or beta 15. From earlier studies, these Rdeoxy signals are attributed to E helix displacement toward the heme in deoxy subunits within R state tetramers, resulting in weakened Trp H-bonds. Asymmetric diligated hybrids, containing both ligands in the same dimer unit, show signals characteristic of the T quaternary contacts, but they are attenuated by 40%. An equilibrium mixture of T and R state molecules is ruled out by the absence of significantly strong Rdeoxy difference bands. Rather, the spectral attenuation is attributed to weakening of the T state contacts at the alpha 1 beta 2 interface. This interpretation is supported by previous observations that the mutational pattern of free energy perturbations for the asymmetric hybrid is T-like and not that of a T/R equilibrium or an R-like state. The asymmetric hybrid represents a third cooperativity state, T', having a T quaternary arrangement of the subunits but a deformed alpha 1 beta 2 interface, with weakened contacts.
The method of diagonalization in a mixed basis (DIMB) that was published previously (Mouawad.), L. and Perahia D., Biopolymers 33, 599, 1993), allows the computation of the low-frequency vibrational modes for large macromolecules. Improvements to this method are presented here, namely the single and double truncation window techniques. The best convergence rate is obtained with the double truncation windows, which couple most efficiently the parts of the macromolecule which are far in sequence but close in space. Both methods were applied to the T-state of hemoglobin, to compare their efficiency. The resulting modes are analyzed in order to study the pathways between T- and R-states of this protein. They show that the quaternary conformational are mainly due to one mode at 2 cm-1.