Probing Heme Active Sites of Hemoglobin in Functional Red Blood Cells Using Resonance Raman Spectroscopy

Abstract

The UV–vis absorption, Raman imaging, and resonance Raman (rR) spectroscopy methods were employed to study cyanohemoglobin (HbCN) adducts inside living functional red blood cells (RBCs). The cyanide ligands are especially optically sensitive probes of the active site environment of heme proteins. The rR studies of HbCN and its isotopic analogues ( 13 CN – , C 15 N – , and 13 C 15 N – ), as well as a careful deconvolution of spectral data, revealed that the ν(Fe–CN) stretching, δ(Fe–CN) bending, and ν(C≡N) stretching modes occur at 454, 382, and 2123 cm –1 , respectively. Interestingly, while the ν(Fe–CN) modes exhibit the same frequencies in both the isolated and RBC-enclosed hemoglobin molecules, small frequency differences are observed in the δ(Fe–CN) bending modes and the values of their isotopic shifts. These studies show that even though the overall tilted conformation of the Fe–C≡N fragment in the isolated HbCN is preserved in the HbCN enclosed within living cells, there is a small difference in the degree of distortion of the Fe–C≡N fragment. The slight changes in the ligand geometry can be reasonably attributed to the high ordering and tight packing of Hb molecules inside RBCs.

The discovery of berberine erythrocyte-hemoglobin self-assembly delivery system: a neglected carrier underlying its pharmacokinetics

“…Moreover, the 1586 and 1633 cm −1 bands belong to the mode vibration of asymmetric stretching of Cα-Cm (alpha and meso positions of porphyrins and pyrrole ring) (Spiro, 1985 ; Li et al., 1990 ). The band of 1586 cm −1 is oxidation state sensitive, and the band of 1633 cm −1 is sensitive to the spin-state for Fe heme, both of these signals are characteristic bands for BHB in Raman spectra (Dybas et al., 2021 ). These stretching modes were observed to be slightly changed when binding with BBR ( Figure 10 ), indicating that the spin state of the heme iron may be affected by the combinations.…”

Raman Spectroscopic Study of TiO2 Nanoparticles’ Effects on the Hemoglobin State in Individual Red Blood Cells

“…To investigate these effects, noncontact and noninvasive means are essential. For this purpose, Raman spectroscopy is a suitable tool for analysis of the Hb function and its dynamics due to a selective enhancement of the Raman active vibrations in different forms of Hb [3,4,10,[21][22][23]. Additionally, Raman mapping allows for a direct observation of the distribution of Hb in different states.…”

“…Additionally, Raman mapping allows for a direct observation of the distribution of Hb in different states. It has been applied for analyzing the formation of Hb derivatives in RBC under varying conditions [21,24], studying the process of erythrophagocytosis of senescent RBC [23], and for diagnosing malaria [24].…”

An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays

“…[ 81 ] On the contrary, the addition of a 125‐fold molar excess of KCN gave rise to the bis‐cyanide‐heme adduct, with a Soret band at 416 nm and a single Q band at 550 nm (Figure S12A, Supporting Information, purple line), as similar UV–Vis response was observed for the bis‐cyanide complex of natural heme‐proteins. [ 82 ] The absorption of the bis‐cyanide‐heme adduct was employed to evaluate the value of the molar extinction coefficient at 416 nm (ε 416 = 61 754 M −1 cm −1 ). Regarding the experiment with the conjugate samples, aliquots of AuNPs@dAb@FeMC6*a and AuNPs@dAb@HRP (750 µL each) were concentrated by centrifugation (10 000 rpm, 30 min, 4 °C) and the collected precipitate (20 µL) was treated with an aqueous solution of KCN (80 µL, 2.5 × 10 −2 M).…”