Cross-Linking of Proteins by Aldotriose: Reaction of the Carbonyl Function of the Keto Amines Generated in Situ with Amino Groups

Nonreductive modification of proteins with glyceraldehyde forming 2-oxo-3- hydroxy propylated protein is mechanistically analogous to nonenzymic glycation reactions. The latent cross-linking potential of glyceraldehyde as a consequence of the reactivity of the carbonyl function of 2-oxo-3-hydroxypropyl groups of nonreductively modified protein has been now investigated. Reaction of RNase A (0.5 mM) with glyceraldehyde (20 mM) at pH 7.4 and 37 °C for 4 h resulted in the intermolecular cross-linking of the protein, with the concomitant development of a yellow chromophore with two new absorption bands having maxima around 305 and 375 nm. The product exhibited a fluorescence that had excitation and emission maxima around 365 and 450 nm, respectively. The presence of NaCNBH₃during the reaction, which selectively reduces the Schiff base adducts of aldotriose to form 2,3-dihydroxypropyl groups on proteins, inhibited both the cross-linking reaction and the development of the absorption and fluorescence characteristics. The hydroxymethyl group of the aldotriose is not an essential moiety since the cross-linking potential of glyceraldehyde is comparable to that of glyceraldehyde 3-phosphate. The formation of cross-links appears to involve the carbonyl function of the keto amines resulting in the formation of Schiff base adducts (ketimine linkages) as the initial event. Consistent with this, incubation of 2-oxo-3-hydroxypropylated RNase A with [¹⁴C]glycine ethyl ester resulted in the incorporation of the reagent into the protein. The cross-linking reaction was inhibited when the reaction of RNase A with glyceraldehyde was carried out in the presence of amino compounds, such as glycine ethyl ester, ethanolamine, glucosamine, and aminoguanidine. An equimolar amount of aminoguanidine inhibited the nonreductive incorporation of [¹⁴C] glyceraldehyde into RNase A by nearly 85%. The inhibition of the cross-linking reaction by the aminoguanidine and other amino compounds is predominantly a consequence of the inhibition of the nonenzymic glycation of RNase A. The results of the present study demonstrate that the protein cross-linking by aldotriose under physiological conditions is latent and is a consequence of the reactivity of the carbonyl function of keto amines generated in situ with the amino groups of protein.