In our efforts to develop a simple assay for determining the number of free cysteines in a protein, we have focused on the use of p-hydroxymercuribenzoic acid (pHMB). This reagent is advantageous because it has a high affinity for free sulfhydryl groups, yet will form a covalent bond with only one sulfhydryl, in contrast to mercuric ions which have a tendency to form a bridge between two separate sulfhydryl groups. Adduct formation with pHMB exaggerates the mass difference between a cystine and an equivalent pair of cysteines. In some cases, pHMB reacts well with cysteine but in other cases, the derivatization is far from complete, in which case, it is difficult to assess how many free sulfhydryl groups are in the protein. To develop a more reliable assay, we are investigating means by which to drive the pHMB reaction further toward completion. A 100-fold molar excess of pHMB improves the extent of derivatization, but requires removal of the excess before the mixture can be analyzed by MALDI. Furthermore, the multiply derivatized forms of the protein which differ in mass by 321 Da can be difficult to resolve above the 10-kDa level. These investigations will continue with heavier organomercurials and with delayed extraction in MALDI to improve the resolving power.
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