Assessment of Cysteine Reactivity of Human Hemoglobin at Its Residue Level: A Mass Spectrometry-Based Approach

Abstract

In general, the reactivity of cysteine residues of proteins is measured by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α₂β₂) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site-specific reactivities of cysteine residues of Hb were investigated using alkylation kinetics with iodoacetamide (IAM). The observed reactivities corroborated well with the differential surface accessibilities of cysteine residues in the crystal structures of human Hb. The proposed method might be used to investigate cysteine reactivities of all the genetic and post-translational variants of Hb discovered to date. In addition, this method can be extended to explore cysteine reactivities of proteins, irrespective of the presence of chromophores and availability of crystal structures.

Keywords:

• Cysteine reactivity
• 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
• human hemoglobin (Hb)
• iodoacetamide (IAM)
• matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)
• sickle cell Hb

Acknowledgments

We are grateful to the volunteers and patients with sickle cell anemia for providing us with the samples for this study.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

We acknowledge the Department of Science and Technology, Government of India for funding the MS facility at the St. John’s Research Institute. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.