Reaction of glycolaldehyde with rroteins: Latent crosslinking

The Schiff base adducts of glyceraldehyde with hemoglobin undergo Amadori rearrangement to form stable ketoamine structures; this reaction is similar to the nonenzymic glucosylation of proteins. In the present studies the analogous rearrangement of the Schiff base adducts of glycolaldehyde with proteins has been demonstrated. However, the Amadori rearrangement of this Schiff base adduct produces a new aldehyde function, an aldoamine, which is generated in situ and is capable of forming Schiff base linkages with another amino group, leading to covalent crosslinking of proteins. Sodium dodecyl sulfate gel electrophoresis of the glycolaldehyde-RNase A adduct showed the presence of dimers, trimers, and tetramers of RNase A, demonstrating the crosslinking potential of this a-hydroxyaldehyde. The crosslinked products exhibited an absorption band with a maximum around 325 nm and fluorescence around 400 nm when excited at 325 nm. The crosslinking reaction, the formation of a 325-nm absorption band, and the development of fluorescence were prevented when the incubation was carried out in the presence of sodium cyanoborohydride. This finding indicates that the Amadori rearrangement that generates a new carbonyl function is a crucial step in this covalent crosslinking. Glycolaldehyde could be a bifunctional reagent of unique utility because its crosslinking potential is latent, expressed only upon completion of the primary reaction. We have been interested in the reaction of a-hydroxyaldehydes with proteins, especially in view of our observation that glyceraldehyde is an efficient antisickling agent in vitro that acts primarily at the stage of aggregation of sickle cell deoxyhemoglobin (1). Only five of the 24 amino groups per ap dimer of hemoglobin are capable of forming stable ketoamine linkages with glyceraldehyde (2, 3), in a reaction analogous to the Amadori rearrangement of glucosylated hemoglobin (4). Recently, we have found that even though valine-1 of the a-chain readily forms a Schiff base with glyceraldehyde, it is refractory to subsequent Amadori rearrangement (5). On the other hand, valine1 of the f3 chain does form stable ketoamine adducts with glyceraldehyde or with glucose by rearrangement. The lysine E-amino groups of hemoglobin also display selectivity in their pattern of reactivity with glyceraldehyde (2) or glucose (6). Therefore, it is clear that our knowledge of the rearrangementprocess as it applies to proteins is limited with respect to both the mechanistic and physiological consequences of the process. The present manuscript addresses the latter question by using glycolaldehyde, a simple a-hydroxyaldehyde. We have now investigated whether the Amadori rearrangement reaction also would occur with the Schiff base adducts of glycolaldehyde with proteins. The rearrangement of the glycolaldehyde-Schiff base adduct would generate a new aldehyde function, which should have the potential to react with another free amino group of the protein and result in covalent crosslinking (Fig. 1). The present study demonstrates that such a reaction indeed takes place when proteins are incubated with glycolaldehyde in the absence of reducing agents. This approach differs from that for monofunctional modification of proteins by reductive alkylation (7). In that system, a reducing agent is present throughout the reaction; Amadori rearrangement and subsequent crosslinking of proteins is thereby precluded. MATERIALS AND METHODS Reaction of Glycolaldehyde with RNase A. RNase A (Sigma) (0.5 mM) in phosphate-buffered saline, pH 7.4, was incubated at 370C for the indicated period at a given concentration of glycolaldehyde (Sigma). At the end of the reaction period, the sample was desalted by passage through a column (2.2 x 30 cm) of Sephadex G-25 (Pharmacia), equilibrated and eluted with 0.1 M acetic acid. The protein, isolated by lyophilization, is referred to as glycolaldehyde-RNase A adduct. Where indicated, the glycolaldehyde adducts were reduced with sodium borohydride as described for the reaction of glyceraldehyde with hemoglobin (1, 2). Reaction of Glycoaldehyde-RNase A Adduct with 2,4-Dinitrophenylhydrazine. This reaction was carried out essentially as described by Fields and Dixon (8). The RNase adduct (0.25 ,Amol) was treated with 1.5 ml of 25 mM 2,4-dinitrophenylhydrazine in 1 M HCl at room temperature for 15 min. The derivatized protein was isolated from the excess reagent by gel filtration on Sephadex G-25, equilibrated and eluted with 0.1 M acetic acid. NaDodSO4/Polyacrylamide Gel Electrophoresis. The extent of crosslinking of RNase A was analyzed by NaDodSO4/ polyacrylamide gel electrophoresis at pH 8.0 on gradient slab gels of 10-20% acrylamide. The stacking gel was made with 4% acrylamide in Tris-HCl buffer, pH 6.8, 10% in NaDodS04. Generally, the amount of protein was of the order of 20 ,ug. The gels were routinely run at 7.5 mA for 1 hr to load the protein and the electrophoresis was carried out for another 1.5 hr at 15 mA in Tris/glycine, pH 8.0, 1% in NaDodSO4. The protein bands were stained with Coomassie blue and destained by washing first with methanol/acetic acid/water, 40:10:50 (vol/ vol), and then with 7% (wt/vol) acetic acid. Amino Acid Analysis. To determine the extent of reaction of the lysine residues with glycolaldehyde, the adduct was reduced with sodium borohydride as described earlier for the reaction of glyceraldehyde with hemoglobin (2). The reduced adduct was dialyzed against water and lyophilized. The samples were hydrolyzed in 6 M HCl under reduced pressure for 22 hr and analyzed on an amino acid analyzer of the design of Spackman et aL (9). Fluorescence Measurements. These determinations were * A preliminary report of these results has been published (25). 3590 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 80 (1983) 3591 HC=N-Protein + H2N-Protein _ s CH20H