Structure of Sickle Cell Hemoglobin Fibers Probed with UV Resonance Raman Spectroscopy

Abstract

The structure of sickle cell hemoglobin (Hb S) (β6 Glu → Val) fibers was probed using UV resonance Raman (UVRR) spectroscopy. For these studies a functional analogue of Hb S, fluoromet Hb S, was used to study structural changes that accompany fiber formation. The amide backbone and aromatic residues of Hb S were selectively investigated using excitation wavelengths of 210, 215, and 230 nm. In the 210 and 215 nm excited fiber spectra, the intensity of all Phe bands increases dramatically. At the excitation wavelengths used, the Phe signal intensity reflects the local environment and increases linearly with increasing ethylene glycol concentration. Thus, UVRR fiber spectra are suggestive of an increase in hydrophobicity of the Phe local environment, which results from the formation of lateral and axial fiber contacts that are primarily nonpolar and hydrophobic in nature. The observed UVRR signal is assigned to the β185 Phe residue, which, together with the β188 Leu residue, forms a hydrophobic lateral contact with the mutated β26 residue. In addition, 230 nm difference spectra are suggestive that H-bonds stabilizing the α1β2 interface are stronger in fibers than in unassociated T-state tetramers. The W3 mode in fiber difference spectra occurs at 1550 and 1565 cm-1, indicative of an increase in Trp spectral heterogeneity. The +6 cm-1 upshift of the W3 mode is attributed to increased hydrophobicity of Trp local environment and is assigned to the β215 Trp residue. Other structural changes include an increase in disorder upon fiber formation, as shown by the frequencies of protein backbone amide vibrational modes. UVRR spectroscopic results are consistent with the structural details of the Hb S double strand observed crystallographically and provide new information regarding local environment and strength of H-bond interactions.

The role of β93 Cys in the inhibition of Hb S fiber formation

“…Thiol modification of hemoglobin was accomplished by 2 different procedures, hereafter referred to as NEM low (NEM-L) and NEM high (NEM-H). Hemoglobin was reacted with NEM at a 2:1 NEM:Hb ratio for 3 h in the NEM-L modification method [33,34], and a NEM:Hb ratio of 5:1 for 4 h in the NEM-H method [37]. The concentration of protein ranged from 1.5 mM Hb to 3.5 mM Hb.…”

“…Sample position was adjusted every 15 min, and the entire sample volume was changed after 1 h of laser exposure. The Phe band occurring at 2020 cm − 1 , was used to perform spectral subtractions, as previously described [34]. Data manipulation and analysis were performed using GramsAI spectral analysis software (ThermoGalactic, Salem, NH).…”

“…1) is dominated by Phe and Tyr ring-stretching modes in the 1585-1614 cm − 1 spectral region [34,57]. In the 215 nmexcited T-R difference spectra, negative peaks are observed at 1605 and 1585 cm − 1 , which correspond to the Y8a, F8b and F8a vibrational modes of Phe and Tyr residues (Supporting information: Fig.…”

“…The structure of the NEM-modified Hb is analyzed using UV resonance Raman spectroscopy, which has been used extensively to characterize the structure of Hb quaternary states and Hb S fibers [33,34]. The choice of UVRR spectroscopy stems from the fact that it has proven to be an extremely versatile method for studying Hb structure and dynamics.…”

“…Spiro and coworkers have previously identified spectral features diagnostic of the critical α 1 β 2 interface [33], which are monitored as a function of quaternary state and fiber formation. In addition, Phe signals, reflective of local environment, increase in intensity with fiber formation, and are used as monitors of the fiber state [34,35]. Thus, one advantage of UVRR spectroscopy over other spectroscopic techniques is the ability to selectively probe both the α 1 β 2 subunit interface, and the intermolecular 1 β 1 -2 β 2 interaction that promotes Hb S fiber formation.…”