Studies on heme proteins using the n.m.r. halide-ion probe technique

Abstract

The n.m.r. halide probe technique is shown to be a sensitive method for detecting the binding of Cl− and Hg2+ to proteins. The direct binding of chloride ions to proteins has been demonstrated for the first time by this technique. There appears to be only a small amount of chloride binding to horseradish peroxidase and to its apoprotein in basic aqueous solutions, but the binding increases markedly as the pH is lowered, indicating that the chloride is binding nonspecifically to positively charged regions on the protein. HgCl2 was found to bind to sperm whale myoglobin in increasing amounts as the pH was raised above 7, indicating that it may be binding to regions of negative charge on the protein. Use of the halide-ion probe technique as a means of detection of reactive sulfhydryl groups by HgCl2 titration led to the finding that there is one reactive sulfhydryl group in peroxidase, and that the rotational correlation time for the peroxidase–HgCl2 complex may be caused by rotation of the segment of the protein containing the sulfhydryl group rather than rotation of the entire protein. One sulfhydryl group was detected in bovine hemoglobin and possibly a second sulfhydryl group of lower activity.