THE ABSORPTION SPECTRA OF HEMOGLOBIN AND ITS DERIVATIVES IN THE VISIBLE AND NEAR INFRA-RED REGIONS

Abstract

Since the pioneering investigations of Vierordt and of Hiifner and his group, numerous reports on the absorption spectra of hemoglobin and its derivatives have appeared in the literature. A detailed summary of these reports is to be found in Heilmeyer’s monograph (1). For the most part these investigations have been confined to the visible portion of the spectrum, although in a few cases the observations were extended into the ultraviolet,. The near infra-red, however, has been singularly neglected, despite the fact that as early as 1914 Hartridge and Hill (2) published some qualitative results indicating the presence of an interesting oxyhemoglobin band in this region. In this laboratory, it became necessary to develop a rapid.and accurate spectroscopic method for the estimation of carbon monoxide in blood which could be adapted to a simple portable instrument. The visible portion of the spectrum proved unsuitable for this purpose, since the character of the oxyhemoglobin and carbonylhemoglobin bands in this region require the use of narrower spectral regions than can conveniently be isolated in such an instrument. The infra-red spectra were investigated in the hope that more suitable absorption bands might be found. The spectrophotometric data for the various hemoglobin derivatives which may be found in the literature are characterized by discrepancies with respect to the absolute values of the absorption coefficients, depending upon the hemoglobin preparations examined, the analytical methods used, and the dispersing power of the spectrophotometers employed by the various investigators. Since the precision of any spectrophotometric method of analysis depends upon the accuracy with which the corresponding absorption coefficients are known, a redetermination of these coefficients was undertaken. The objectives of this investigation were to determine as accurately as possible the absorption coefficients of various hemoglobin derivatives in whole hemolyzed human blood and to compare these with constants obtained with pure hemoglobin, in order that the contribution of hemoglobin to the absorption of whole blood might be evaluated. A similar study was made of the spectra of these substances in the near infra-