Real Time Monitoring of Sickle Cell Hemoglobin Fiber Formation by UV Resonance Raman Spectroscopy

Abstract

In sickle cell hemoglobin, individual tetramers associate into long fibers as a consequence of the mutation at the beta6 position. In this study UV resonance Raman spectroscopy is used to monitor the formation of Hb S fibers in real time through aromatic amino acid vibrational modes. The intermolecular contact formed by the mutation site ((1)beta(1)6 Glu-->Val) of one tetramer and the (2)beta(2)85 Phe-(2)beta(2)88 Leu hydrophobic pocket on a different tetramer is observed by monitoring the increase in signal intensity of Phe vibrational modes as a function of time, yielding kinetic progress curves similar to those obtained by turbidity measurements. Comparison of individual spectra collected at early time points (<1000 s) show small Phe intensity changes, which are attributed to weak transient associations of Hb S tetramers during the initial stages of the polymerization process. At later times (1000-2000 s) Phe signal intensity steadily increases because of increasing hydrophobicity of local Phe environment, a consequence of forming more stable (1)beta(1)-(2)beta(2) contacts. Tyr and Trp vibrational modes monitor H-bond strength between critical residues at the alpha(1)beta(2) interface of individual tetramers. Kinetic progress curves generated from these signals exhibit two distinct transitions at 2040 and 7340 s. These transitions, which occur later in time than those detected either by turbidity (1560 s) or by Phe signal intensity (1680 s), are attributed to initial fiber formation and subsequent formation of larger assemblies, such as macrofibers or gels. These results provide molecular insight into the interactions governing Hb S fiber formation.