Development of sensitive metalloporphyrin probes for chemiluminescent imaging detection of serum proteins

Abstract

The development of metalloporphyrin- (ferric protoporphyrin IX chloride (FePP), cobalt (III) protoporphyrin IX chloride, copper (II) protoporphyrin IX) enhanced chemiluminescent (CL) imaging detection of serum proteins after PAGE is described in this article. The detection is based on the catalytic activity of metalloporphyrins, especially FePP, in the CL reaction of the luminol-H2O2 system. Some relatively low abundant proteins such as hemopexin (Hpx) and complement C4 are sensitively detected by FePP-enhanced CL imaging. Other proteins such as haptoglobin, apolipoprotein A-1, complement C3, and alpha-1-antitrypsin are also detected and identified by MS and MS/MS techniques. Detection limit of Hpx is as low as 20 ng, without the need of expensive antibodies or tedious immunoassay procedures. The mechanism of the proposed method is discussed employing standard proteins. The application to the analysis of different protein patterns in healthy people and in Thalassemia patients is being investigated.

Hemin Binds to Human Cytoplasmic Arginyl-tRNA Synthetase and Inhibits Its Catalytic Activity

“…For many heme-containing proteins, the heme moiety facilitates the binding of diatomic gases, such as NO, CO, and O 2 (20) . Soluble guanylate cyclase is a heme sensor protein that selectively binds NO at the heme iron, triggering the enzyme to convert GTP to cGMP (35). Heme-regulated eIF2␣ kinase (HRI) regulates protein synthesis in red blood cells, and its kinase activity is strongly inhibited by hemin (22,23).…”

Variables That Influence Cellular Uptake and Cytotoxic/Cytoprotective Effects of Macrocyclic Iron Complexes

“…It relies on the emission of blue light in the luminol/H 2 O 2 reaction, resembling routinely employed methods in biochemical research and forensic detection that are based on horseradish peroxidase and hemoglobin, respectively . The prosthetic group responsible for catalysis in these biomolecules is iron(III) protoporphyrin IX, and synthetic iron(III) porphyrins have been used for the same purpose in purely chemical systems . The potency of the iron(III) corrole 1-Fe toward the luminol/H 2 O 2 reaction is identified here for the first time.…”

“…We have demonstrated significant differences between the cellular accumulation of iron(III) macrocycles of different polarity and shown that both cytotoxicity and cytoprotection properties vary very much within the examined series. Determination of cell accumulation was achieved using a very simple, rapid, and sensitive detection method, which may, in principle, be applied for any cell type and for other compounds that catalyze the oxidation of luminol . Of the four examined iron complexes, only the amphipolar corrole displays all desired features that are essential for utilization as a cell-protecting antioxidant: sufficiently large cellular uptake, nontoxicity toward the cells, and intracellular catalytic activity against the most important ROS and RNS.…”