Magnetophoretic and spectral characterization of oxyhemoglobin and deoxyhemoglobin: Chemical versus enzymatic processes

Abstract

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

Applications of magnetic and electromagnetic forces in micro-analytical systems

“…The population shifted to a lower velocity, indicating the loss of Hb in RBC due to aging. In addition, they observed iron storage in circulating monocytes, 34,35 the relation between phosphatidylserine expression and magnetic mobility, 36 enzymatic deoxidation of Hb, 37 iron transport in cancer cells, 38 and showed the possibility of hematology analyzer detection. 39,40…”

Iron in blood cells: Function, relation to disease, and potential for magnetic separation

“…SCA is an autosomal recessive disorder caused by a mutation in the β-globin gene of the Hb. In this mutation, the substitution of glutamic acid by valine occurs, resulting in the production of sickle Hb (HbS) molecules, which interact with adjacent HbS molecules under deoxygenation conditions and create HbS polymers (M. R. H. Weigand et al, 2021). These aggregates form lengthy chains, distorting the RBC into a sickled shape and impeding the cell's passage through blood vessels (Lonergan et al, 2001).…”

“…Under this scenario, the use of magnetophoresis can be a cheaper solution to diagnose anemias since the introduction of powerful and low‐cost neodymium magnets and modern computer imaging technologies enable the development of instruments that can determine the magnetic susceptibility of blood cells, which are related to their Hb and iron content (Chalmers et al, 1999, 2017). For example, recent works have suggested the use of a permanent magnet‐based microsystem referred to as cell tracking velocimetry (CTV) (Chalmers et al, 1999; Kim, Gómez‐Pastora, Gilbert, et al, 2019; Kim, Gómez‐Pastora, Weigand, et al, 2019; R. H. Weigand et al, 2021) as a portable and inexpensive hematology analyzer able to measure traditional RBC indices and novel indices such as the percentage of hypochromic RBCs and the red cell distribution width (Gómez‐Pastora, Weigand, et al, 2022).…”

Potential of cell tracking velocimetry as an economical and portable hematology analyzer

“…We have previously developed a system, referred to as the cell tracking velocimetry (CTV), to measure the mass of Hb in fresh RBCs on a cell by cell basis using principles from the pioneering work of Pauling and Coryell studying the magnetism of Hb in different chemical states 17 , 18 . CTV uses a combination of a microscope camera and a microfluidic channel within a well-defined magnetic energy gradient to track the movement of cells and particles under the direct influence of magnetic and gravitational fields 19 – 21 . Mathematically, the magnetically and gravitationally induced velocities, u m and u s , can be described as follows: where the subscripts cell and fluid refer to the cell and suspending fluid, χ is the magnetic susceptibility, ρ is the density (1,100 kg/m 3 ), D and V are the diameter and volume of a cell (particle), η is the viscosity of the suspending fluid (9.7*10 –4 Pa*s), and g is the acceleration due to gravity (9.8 m/s 2 ).…”