Heme-CO religation in photolyzed hemoglobin: a time-resolved Raman study of the Fe-CO stretching mode.

Time-resolved resonance Raman spectroscopy has been employed to monitor geminate heme-CO rebinding in photolyzed HbCO. The excitation frequency was tuned to enhance the scattering from rebound heme sites 20-500 ns subsequent to CO photolysis. The behavior of vFe-C during ligand rebinding has important ramifications concerning heme pocket dynamics of the distinct equilibrium configurations of the six-coordinate heme sites. During the geminate phase of recombination, the Fe-CO bond strengths and configurations of the rebound sites (inferred from the positions and line widths of vFe-C) were found to be the same as those of equilibrium configurations of HbCO within 500 ns of CO photolysis for all samples. No evidence was found for the existence of transient metastable configurations during geminate recombination. Spectra obtained at earlier times (100 ns) revealed small differences in the geminate rebinding rates of the two equilibrium configurations. Since there is little or no further CO rebinding between 100 and 500 ns after photolysis, some interconversion must occur between the dominant HbCO configurations on a submicrosecond time scale.

Mapping gold-labeled receptors on cell surfaces by backscattered electron imaging and digital image analysis: studies of the IgE receptor on mast cells.

The response of cells to signaling molecules such as hormones, growth factors, and immune mediators that bind to cell-surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL-2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold-conjugated probes that bind directly or indirectly to the IgE-receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold particles.