Allylation of intraerythrocytic hemoglobin by raw garlic extracts

Abstract

Recent studies have shown that deoxygenated human red blood cells (RBCs) converted garlic-derived polysulfides into hydrogen sulfide, which in turn produced vasorelaxation in aortic ring preparations. The vasoactivity was proposed to occur via glucose- and thiol-dependent acellular reactions. In the present study, we investigated the interaction of garlic extracts with human deoxygenated RBCs and its effect on intracellular hemoglobin molecules. The results showed that garlic extract covalently modified intraerythrocytic deoxygenated hemoglobin. The modification identified consisted of an addition of 71 atomic mass units, suggesting allylation of the cysteine residues. Consistently, purified human deoxyhemoglobin reacted with chemically pure diallyl disulfide, showing the same modification as garlic extracts. Tandem mass spectrometry analysis demonstrated that garlic extract and diallyl disulfide modified hemoglobin's beta-chain at cysteine-93 (beta-93C) or cysteine-112 (beta-112C). These results indicate that garlic-derived organic disulfides as well as pure diallyl disulfide must permeate the RBC membrane and modified deoxyhemoglobin at beta-93C or beta-112C. Although the physiological role of the reported garlic extract-induced allyl modification on human hemoglobin warrants further study, the results indicate that constituents of natural products, such as those from garlic extract, modify intracellular proteins.

FIG. 1.

Garlic extract modifies human beta-hemoglobin (Hb). Freshly prepared human red blood cells were treated with either (A) water or (B) garlic extract. Hb was purified from the treated red blood cells and subjected to BioMass analysis. (A) Hb purified from control samples showed two major peaks at 15,130.5 and 15,870.5, corresponding to alpha- and beta-chain, respectively. (B) In contrast, Hb purified from garlic extract-treated samples showed a mass shift of the peak corresponding to beta-chain. The hemoglobin beta-chain of the garlic extract showed a molecular mass of 15,941.5, a difference of 71 atomic mass units. The spectra are representative of at least three independent experiments. NL, normalization level; +c NSI Full ms, positive mode nanospray ionization full mass spectra.

FIG. 2.

Garlic extract modifies human beta-Hb inside red blood cells. Freshly prepared human red blood cells were treated with (A) vehicle, (B) garlic extract alone, or (C) iodoacetamide (IAA)/garlic extract. The purified Hb proteins from all samples were subjected to BioMass analysis. (A) Control sample showed peaks corresponding to unmodified Hb alpha- and beta-chains. (B) The purified Hb protein from garlic extract-treated samples showed the mass shift in the beta-chain, as previously detected. (C) The purified Hb from the IAA-pretreated samples showed the alpha-chain at a mass similar to the control, but the beta-chain showed a mass shift of 53 atomic mass units compared to control samples, indicating that the membrane-permeable IAA modified human beta-Hb and interfered with garlic extract-induced modification. The mass spectra are representative of at least three independent experiments.

FIG. 3.

Diallyl disulfide modifies human Hb beta-chain. Freshly prepared human red blood cells were treated with either (A) vehicle or (B) diallyl disulfide for 18 hours at room temperature. Hb was purified from the samples and subjected to BioMass analysis. (A) The control sample, consistently, showed the same mass for both alpha- and beta-chains of Hb. (B) The diallyl disulfide-treated sample showed a mass shift for the beta-chain of 71 atomic mass units, similar to the one detected for the samples treated with garlic extract. A trace of unmodified beta-chain was still detected after 18 hours of incubation (arrow). The results suggest that diallyl disulfide induced the formation of a S-allyl-cysteine adduct on the beta-chain molecule (inset).