Abstract

The structure of proteins is defined by two main types of covalent bonds; the peptide bonds that link the amino acid residues and disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds are introduced during protein folding and their formation is assumed to be complete in the mature, functional protein. We tested this assumption by quantifying the redox state of disulfide bonds in human blood proteins in their native environment. Using a differential cysteine alkylation and mass spectrometry method, we measured the redox state of disulfide bonds in circulating fibrinogen and von Willebrand factor. There is an extraordinary disulfide lability in the proteins, with 27 bonds in the two proteins ranging from 3 to 50% reduced in healthy human donors. Modelling of the data indicates that the proteins exist in hundreds of different disulfide-bonded states in the circulation. Different covalent states of fibrinogen are associated with different binding activities and their distribution is changed by fluid shear forces and altered in patients with cardiovascular disease, indicating that the different states have different functions and are dynamic. These findings have implications for protein function generally and how proteins are targeted in experimental settings and for therapeutic purposes.