Post-Amadori Reactions of Serum Proteins

Cochrane, Sheila (1995). Post-Amadori Reactions of Serum Proteins. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fb5c

Abstract

The formation of glycofluorophores and the fragmentation of proteins is only seen if glucose (or another reducing sugar) is included in the incubation. Although similar changes have been reported in the absence of sugars, it has been shown here that these changes are glucose dependent. Contrary to previous work, I have shown that the presence of bound lipid on HSA does not alter the formation of Amadori product. Fragmentation of HSA is also unaffected by the presence ofbound lipid, but the formation of fluorescence by native HSA is 2.5 times greater than that of delipidated HSA. This shows that (i) tire enhanced fluorophore formation seen in native HSA is independent of the amount of Amadori product, (ii) that at least two con^onents of fluorescence on native HSA exist and (iii) that the mechanisms of fragmentation and lipid-dependent fluorescence formation are not the same.

Both fragmentation and fluorescence formation were inhibited by the transition-metal chelator DTPA, low ing that both these reactions are transition-metal catalysed. The formation of glycofluorophores appears to be partially dependent on the high affnity binding of transition metals to the protein, but the glucose-induced fragmentation of both albumin and human transferrin occurs only when the transition metal species present is copper. No fragmentation of the iron binding protein transferrin is seen when iron is included in the incubations in the place of copper. Both these reactions were also inhibited by the use of HEPES buffer in the place of phosphate buffer. HEPES buffer, unlike phosphate buffer, has free radical scavenging properties so therefore it would appear that in addition to being transition-metal catalysed, these reactions are free radical mediated processes. The carbonyl scavenger, aminoguanidine, inhibited the production of glycofluorophores implicating a carbonyl intermediate in this reaction. Pre-incubation of glucose with copper caused an increase in the amount of fluorescence initially observed indicating that autoxidation of glucose plays a role in fluorophore formation, although it is not possible to say whether this is a major reaction pathway or not.