Demonstration of haemoglobin associated with isolated, purified spectrin from senescent human red cells

Abstract

Employing a direct and sensitive radioimmunoassay (RIA) we have confirmed the presence of haemoglobin associated with isolated, purified spectrin from senescent red cells. Haemoglobin associated with spectrin occurs in the highest amount in cells with an MCHC greater than 36 g/dl and is approximately 3% of the total spectrin extract. Spectrin from the young cells had the least haemoglobin, while an intermediate amount was found in unfractionated, whole red cells. The RIA results were in close approximation with estimation of the haemoglobin-spectrin complex obtained by carefully integrating the Coomassie blue stain profiles from 4% SDS PAGE in densitometric scans from isolated spectrin.

Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes

“…This analysis also demonstrated that h1 is completely extractable from cell ghosts, whereas h2 is only 50% extractable. These findings are incorporated into a hypothesis linking globin-spectrin complexation and the consequent inhibition of spectrin dimer self-association to the clustered band 3 induced by peroxide, a finding that is consistent with the established role of Hb oxidation as the initial step in the production of oxygen free radicals in RBCs. "'…”

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

“…It is well known that RBCs are densely packed with large amounts of hemoglobin. Hemoglobin is also bound to the membrane, as has been determined after several washes [40]. The absorption spectrum of hemoglobin originates from heme, having an intense Soret or B-band (~400-430 nm, depending on oxidation state) and weak transition to the Q-band (~550nm).…”

“…9(b) tracks absorption at 414nm with increasing washes and shows that a certain percentage of hemoglobin remained. Previous methods with subsequent washing of RBCs also found a small percentage of hemoglobin in the membrane that cannot be removed via washing [40]. …”

Crosstalk between Red Blood Cells and the Immune System and Its Impact on Atherosclerosis

“…RBC oxidation may be involved in RBC aging and oxidized RBCs show the phenotype of senescence. The major feature of the senescent RBCs is the clustering and/or the breakdown of Band 3 including the binding of oxidized Hb to high affinity sites on Band 3 [ 30 , 31 ], and the complexation of Hb with spectrin is also a prominent and probably prior marker of in vivo RBC aging process, tightly correlated with increased RBC rigidity, decreased deformability, echinocytosis, and erythrophagocytosis [ 32 , 33 ]. Considering that Band 3 modifications and modification of preexisting molecule generating pathways are mostly the downward consequence of oxidative provocations, it is likely that in conditions of distorted homeostasis oxidized RBCs result as product of an “accelerating senescence” pathology associated with the premature appearance of cellular senescence phenotype in younger RBCs.…”