Ligand‐Linked Changes of Ultrasound Absorption of Hemoglobin

Abstract

The acoustic absorption of hemoglobin solutions at 1.88 MHz has been studied in dependence of ligand binding and pH. In the physiological pH range the excess absorption of oxyhemoglobin is increased compared to deoxyhemoglobin. Evidence is presented that in human hemoglobin the additional absorption of oxyhemoglobin is caused by a proton transfer relaxation process involving the a chain N-terminal a-amino groups. This process cannot be observed in deoxyhemoglobin because of fixation of the N-terminal a-amino groups in a salt bridge. Further investigations of carbonmonoxy, nitrosyl, aquomethemoglobin and fluoromethemoglobin in the absence and presence of inositol hexakissulfate show that acoustic absorption measurements offer an additional tool to characterize in greater detail the different conformational states of hemoglobin.The study of conformational changes in the globin part of the hemoglobin molecule, associated with ligand binding, is a central problem of hemoglobin chemistry. In recent years increasing use has been made of relaxation methods, which have significantly increased knowledge about the dynamical aspects of conformational equilibria. A major problem associated with the use of relaxation methods in protein chemistry is that the complexity of the protein structure very often makes it difficult to assign to an observed signal change a specific type of relaxation process or identify the specific groups involved. This is especially the case with ultrasound methods, where hemoglobin has served as a model protein for analysis of specific sound-absorbing processes in several studies. A satisfactory explanation for processes causing excess sound absorption in the MHz frequency range has been advanced only for the absorption maxima found in the very acidic (pH < 3.5) and alkaline (pH > 11) ranges. There the excess sound absorption seems to be caused predominantly by dissociative proton transfer reactions of the basic and acidic amino acid sidechains [I, 21. Processes responsible for sound absorption in the pH range between 6 and 9, i.e. the physiologically important pH range, are less well established. There are, however, several results pointing towards an important role of direct internal proton transfers between adjacent amino acid side-chains as contributors to excess sound absorption at neutral pH. Thus, White and Slutsky [3] have computed that in bovine hemoglobin one internal direct proton transfer Abbreviations. Hb, hemoglobin; Mb, myoglobin. per aP dimer could theoretically account for up to 25 % of the total acoustic excess absorption observed at neutral pH [3].With one exception ultrasound studies on hemoglobin have not been concerned with the question of whether a difference in ultrasound absorption exists between oxy and deoxyhemoglobin. The work of Schneider et al.[4] on bovine hemoglobin gave no conclusive evidence about significant differences in ultrasound absorption of oxy and deoxyhemoglobin. The present study investigates ligand-dependent changes in excess acoustic absorption of he...

Protein Diffusion in Living Skeletal Muscle Fibers: Dependence on Protein Size, Fiber Type, and Contraction

“…Insoluble material was then removed by centrifugation (2000 ϫ g for 20 min at 4°C). Human HbA was prepared according to the method of Jürgens et al (1980) from donated blood received from the local blood bank. Earthworm (Lumbricus terrestris) hemoglobin was purified according to the method of Gros (1978).…”

Age-Dependent Acoustic and Microelastic Properties of Red Blood Cells Determined by Vector Contrast Acoustic Microscopy

“…Typically, attenuation increases with increased cellular hemoglobin concentration (Schenk et al, 1988) and with cross-linking (Kremkau et al, 1973) in RBCs, which would favor an increase in attenuation with advanced age. However, the attenuation of ultrasonic waves by hemoglobin has been shown to exhibit a pronounced increase in the presence of organic phosphates, namely 2,3-disphosphoglycerate, which, on the other hand, tend to diminish in older RBCs to less than a third of the amount detected in younger RBCs (Jürgens et al, 1980; Fornaini et al, 1985). Such factors may account for the observed values of the attenuation coefficient, which do not correlate monotonically with age.…”