Molecular interactions of re‐released proteins in electrophoresis of human erythrocytes

Abstract

Recently, we found that hemoglobin (Hb) could be re-released from live erythrocytes during electrophoresis release test (ERT). The re-released Hb displays single-band and multiple-band re-release types, but its exact mechanism is not well understood. In this article, the protein components of the single-band re-released Hb were examined. First, the re-released band of erythrocytes and the corresponding band of hemolysate, which was used as control, were cut out from starch-agarose mixed gel. Next, proteins were recovered from the starch-agarose mixed gel by freeze-thaw method. After condensing in a vacuum freeze drier, the samples were loaded onto a 5-12% SDS-PAGE. After electrophoresis, three protein bands (16, 28.9, and 29.3 kDa) emerged from the erythrocytes re-released Hb single-band (R-R), but only one band (29.3 kDa) emerged from the corresponding hemolysate control band (H-R). Finally, these bands were analyzed by MALDI-TOF MS. The results showed that these proteins were beta-globin (16 kDa), carbonic anhydrase 1 (CA1, 28.9 kDa), and carbonic anhydrase 2 (CA2, 29.3 kDa). Because CA2 exists in both erythrocytes re-released band and hemolysate control band, we conclude that the single-band re-released Hb is mainly composed of HbA and CA1. Studying the possible interaction between HbA and CA1 will help us further understand the in vivo function of Hb.

Abnormal increased re-released Hb from RBCs of an intrahepatic bile duct carcinoma patient was detected by electrophoresis release test

“…The increased Hb re-release was firstly observed in ȕ-thalassemia patient [1], and then it was observed in diabetes patients and some general surgical patients [3]. To the contrary, the re-released Hb could also decrease distinctively or disappear from RBCs of hereditary spherocytosis patient.…”

“…Hemolysate was prepared by continuously adding 200 μL saline and 100 μL CCl 4 to the RBCs. After turbulent mixing, the sample was centrifuged at 12 000 rpm for 10 minutes and the upper red hemolysate was pipetted out carefully and stored at 4 for later use [1][2][3]. The 2% starch-agarose mixed gel (4:1) was prepared with TEB buffer (pH 8.6) as described previously [1].…”

“…As to the mechanism of ERT, we primarily speculate that the electric pulse from turning-on and -off the power supply could create plasmatorrhexis of RBCs. Hb, existing free in the cytoplasma, would be released during the first cycle of electrophoresis; while the other Hb, binding with the RBC membrane, would be released during the subsequent cycle of electrophoresis [3]. The amount of re-released Hb may be associated with the status of both Hb and RBC membrane, which can be affected by many diseases, such as hematonosis, tumor, diabetes and so on [4][5][6][7][8].…”

“…Generally, little re-released Hb can be observed during ERT. Our previous experiments proved that the re-released Hbs were increased distinctively from RBCs of ȕ-thalassemia, diabetic and some general surgical patients [3]. As we known, oxidative damage is the important factor to destroy the RBC membrane [9] and membrane-bound Hb is an important marker of oxidative injury in RBCs [10].…”

Comparative Study of the Amount of Re-released Hemoglobin from α-Thalassemia and Hereditary Spherocytosis Erythrocytes

“…To our surprise, another new Hb band was found to be released from the origin, which was named "single-band re-release" as opposed to the "initial release". When the power outages were simulated more than once, multiple Hb bands would appear between HbA and origin, and this phenomenon was named as multiple-band re-release or ladder-band re-release [13]. Based on these experiments, isotonic and hypotonic HRT and double-dimensional HRT were developed subsequently.…”