Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser72, beta Thr84, beta Thr87, beta Ser89, beta His92, beta Cys93, beta Cys112, beta Thr123, and beta His143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with alpha His45, alpha Tyr42, beta Cys93, beta His92, and beta Ser72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser44, beta Thr84, and beta His92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at alpha His20, alpha Tyr24, alpha Tyr42, alpha His45, beta Ser44, beta Thr84, and beta Ser89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.