Dependence of visible spectrum [epsilon (lambda)] of fully oxygenated hemoglobin on concentration of hemoglobin

Lambert-Beer's law assumes that the extinction coefficient of a dye is not influenced by its concentration. Some experiments of Barnikol (Proc. Int. Congr. Physiol. Sci. 27th Paris 1977; Respiration 36: 86-95, 1978) led to the presumption that especially at highly concentrated hemoglobin (Hb) solutions Lambert-Beer's law does not hold. To further elucidate this problem we have measured the extinction coefficient of completely oxygenated hemoglobin (epsilon HbO2) over a wide range of Hb concentrations (CHbO2). Our results prove clearly that epsilon HbO2 in visible range as well as in the Soret region depends on CHbO2. In our opinion the invalidity of Lambert-Beer's law is caused by the self-association of the Hb molecules. This concept is a powerful tool to explain the high n values (n greater than 4) which were obtained by some authors in animal blood (bird, frog, trout, earthworm). Furthermore the influence of the self-association on the physicochemical properties of concentrated solutions of human hemoglobin is discussed.