The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, Nε-(2,3-dihydroxypropyi)lysine, is a substituted α-amino alcohol. This α-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its Nε-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal Nε-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraidehyde used. Periodate oxidation of dihydroxypropylated ribonuclease A regenerated the full enzymic activity of the parent protein. These results demonstrate that dihydroxypropylation of amino groups is a valuable addition to the arsenal of reversible amino group modification procedures.
|Original language||English (US)|
|Number of pages||7|
|Publication status||Published - 1987|
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