Conformational studies on modified proteins and peptides. IX. Conformation and immunochemistry of hemoglobin reduced at some carboxyl groups by diborane.

A derivative of apohemoglobin was prepared by reduction with diborane at -10 degrees C for 2 h. Four aspartate and two glutamate side chains were reduced per half molecule to their corresponding alcohols. The modifications were identified as: alpha asp 64, alpha-asp 74, alpha-asp 75 and alpha asp 85;beta-glu 121 and beta-glu 6 (or beta-glu 7). Recombination of reduced apohemoglobin with unmodified ferriheme yielded the corresponding reduced hemoglobin derivative which was electrophoretically homogeneous. Hemoglobin and reduced hemoglobin had different electrophoretic mobilities and showed slight spectral differences. Titration with ferriheme revealed that reduced globin possessed an anomalous heme-binding behavior. The s 20,w of reduced hemoglobin was slightly higher than that of hemoglobin. Agregation of the derivative was also apparent from its behavior in gel filtration. ORD and CD measurements indicated substantial conformational differences between hemoglobin and its derivative. Immunochemical studies showed that the derivative reacted poorly with antisera to hemoglobin and, conversely, hemoglobin showed little reaction with antisera to the derivative. The results are discussed in terms of the three-dimensional structure of hemoglobin in the crystalline state and its relationship to the structure in solution.