Interaction of carbonic anhydrase I released from red blood cells with human plasma in vitro

Abstract

Red blood cells (RBCs) constitute ∼50% of the bloodstream and represent an important target for environmental pollutants and bacterial/viral infections, which can result in their rupture. In addition, diseases such as sickle cell anaemia and paroxysmal nocturnal haemoglobinuria can also result in the rupture of RBCs, which can be potentially life-threatening. With regard to the release of cytosolic metalloproteins from RBCs into the blood-organ system, the biochemical fate of haemoglobin is rather well understood, while comparatively little is known about another highly abundant Zn-metalloprotein, carbonic anhydrase (CA I). To gain insight into the interaction of CA I with human blood plasma constituents, we have employed a metallomics tool comprised of size-exclusion chromatography (SEC) coupled online with an inductively coupled plasma atomic emission spectrometer (ICP-AES), which allows to simultaneously observe all Cu, Fe, and Zn-metalloproteins. After the addition of CA I to human blood plasma incubated at 37°C, the SEC-ICP-AES analysis using phosphate buffered saline (pH 7.4) after 5 min, 1 h, and 2 h revealed that CA I eluted after all endogenous Zn-metalloproteins in the 30 kDa range. Matrix-assisted laser desorption-time of flight mass spectrometry analysis of the collected Zn-peak confirmed that CA I eluted from the column intact. Our in vitro results suggest that CA I released from RBCs to plasma remains free and may be actively involved in health-relevant adverse processes that unfold at the bloodstream-endothelial interface, including atherosclerosis and vision loss.

Keywords: Endothelium; Mechanism of toxicity; Plasma protein binding; Red blood cell lysis; Toxic metals; Zn-metalloprotein.

Graphical Abstract

Toxic metal-induced red blood cell microbursts liberate carbonic anhydrase I which then impinges on endothelial cells thus possibly contributing to the etiology of atherosclerosis.

Fig. 1

Representative Cu, Fe, and Zn-specific chromatograms obtained by SEC-ICP-AES for the analysis of rabbit blood plasma that had been spiked with bovine CA after 5 min, 1 h, and 2 h. Column: Superdex 200 HR SEC column (30 × 1.0 cm ID, 8.6 µm particle size, fractionation range: 600–10 kDa); Mobile phase: PBS-buffer (pH 7.4); Temperature: 20°C; Flow rate: 0.75 mL/min; Injection volume: 500 µL. The retention times of a mixture of molecular weight standards and that of pure bovine CA (30 kDa) are indicated at the top of the figure.

Fig. 2

Representative Cu, Fe, and Zn-specific chromatograms obtained by SEC-ICP-AES for the analysis of human blood plasma that had been spiked with human CA I after 5 min, 1 h, and 2 h. Column: Superdex 200 HR SEC column (30 × 1.0 cm ID, 8.6 µm particle size, fractionation range: 600–10 kDa); Mobile phase: PBS-buffer (pH 7.4); Temperature: 20°C; Flow rate: 0.75 mL/min; Injection volume: 500 µL. The retention times of a mixture of molecular weight standards and that of pure human CA I (30 kDa) are indicated at the top of the figure.

Fig. 3

Representative Zn-specific chromatograms obtained by SEC-ICP-AES for the analysis of rabbit blood plasma that was spiked with bovine CA after 2 h (top) and human blood plasma that was spiked with CA I. Column: Superdex 200 HR SEC column (30 × 1.0 cm ID, 8.6 µm particle size, fractionation range: 600–10 kDa); Mobile phase: PBS-buffer (pH 7.4); Temperature: 20°C; Flow rate: 0.75 mL/min; Injection volume: 500 µL. The retention times of a mixture of molecular weight standards and that of pure bovine CA (30 kDa) are indicated at the top of the figure.

Fig. 4

MALDI-TOF MS identification of the Zn-peak as CA I after SEC-ICP-AES analysis using 50 mM Tris buffer (pH 7.4) as the mobile phase.