Fructation induced cross-linking of beta-lactoglobulin and lysozyme.

Protein glycation (non-enzymatic glycosylation) and the development of advanced glycation end-products (AGEs) is strongly associated with the pathogenesis of diabetic secondary complications [I ] . Hydroxyl radicals generated from superoxide via the Fenton reaction have been implicated for in virro AGE formation [2]. The term glycoxidation has been coined to emphasise the involvement of metal catalysed oxidation in AGE formation [I]. Fructose produces AGEs at a much faster rate than glucose and has been implicated in the pathogenesis of diabetic cataract 131. Fructose is thought to be produced from glucose by way of the sorbitol pathway especially during hyperglycaemia. where intracellular fructose concentrations have been found to equal or even exceed extracellular glucose concentration 141. In the serum of normal healthy individuals the fructose concentration can rise up to 0.4mM 151. The sugar-induced cross-linking of serum proteins to structural proteins is important in the pathogenesis of glomerular basement membrane thickening and diabetic peripheral neuropathy [6.7]. Makita el a1 (1994) have detected low molecular weight (2-6kD) AGE-modified molecules in the serum. These molecules were detected in increasing concentration with loss of renal function and had a high cross-linking potential to collagen [8]. To date, in virro studies of glycation-induced polymerisation have concentrated on single proteins. This study illustrates the fructose induced cross-linking of protein mixtures, which is more relevant to the in vivo scenario. In vitro fructation of 0-lactoglobulin (bovine milk) and lysozyme (hen egg) was carried out at 37OC in 0.1M sodium phosphate buffer, at pH 7.4. over 7 days with 3mM a i d e included to control microbial growth. Fructation of 2Omg/ml protein was performed using IOOmM fructose. The effect of the metal chelator diethylenetriaminepenta-acetic acid (DTPA. ImM) was also investigated. Fluorescence is often used to quantify AGEs. Column 4 of Fig. 1 shows the fluorescence of fructated lysozyme as a percentage of the fluorescence of fructated 0-lactoglobulin (column 5 ) . If this chart were analysed on its own, it would suggest that lysozyme does not form AGEs as rapidly as 0-lactoglobulin. However comparison of the same fructated samples by electrophoresis (see lanes 4 and 5 in Fig.2) shows a similar degree of polymerisation. (lysozyme dimer of 28kD, 0lactoglobulin dimer of 36kD and trimer of 54kD). This illustrates that there are many different routes to AGE formation. The presence of other proteins may enhance the routes to some end-products at the expense of others. Fructation of lysozyme and 0-lactoglobulin together changes both the degree and pattern of cross-linking. Fructation of both proteins together in the same solution produced considerable precipitation: 1 2 3 4 5 6 7 8 9