Heparin-induced structural modifications and oxidative cleavage of human serum albumin in the absence and presence of glucose--implications for transcapillary leakage of albumin in hyperglycaemia.
Both unfractionated and fractionated, low-molecular-mass heparins were tested on human serum albumin in the absence and presence of glucose at concentrations similar to those frequently found in diabetic hyperglycaemic patients, to ascertain whether heparin and glucose interfered with each other in affecting the conformation of albumin. Reproducible results were obtained with both heparins when used at equal masses, but not when used at equal molar concentrations, suggesting a crucial role of the amount of the saccharide units in determining the observed effects. Spectroscopic studies showed that the binding sites of glucose and heparin on albumin do not overlap and that changes in protein structure depend on complex and mutual interference of glucose and heparin with the protein, although the effects of heparin in modifying the chromophore environment and increasing the ordered structure of the protein also prevailed in the presence of glucose. Heparin binding to albumin rapidly gave rise to oxidative reactions, which were responsible for the increase in the carbonyl content of the protein together with its higher susceptibility to tryptic digestion. Glucose enhanced and prolonged the production of heparin-induced oxidants. Oxidation caused peptide bond cleavage at Lys323 in the primary structure of albumin, yielding two large fragments of 27.5 kDa and 35 kDa which aggregated to form disulphide-linked homodimers visible in SDS/PAGE as two new bands of 54 kDa and 74 kDa, respectively. This was accompanied with a reduction in Val, Glu, and Gly residues, only partially counterbalanced by an increase in Thr and Ser residues. While only a small percentage of albumin molecules underwent fragmentation in the presence of heparin with glucose, albumin turned out to display in an even higher proportion structural modifications consistent with a higher degree of ordered structure. The mechanism(s) underlying this heparin-driven effect and possible physiopathological implications in vivo are discussed.
Finotti
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P
Department of Pharmacology, University of Padova, Italy. finotti@dfem.unipd.it
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D002199:Capillary Permeability; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D006943:Hyperglycemia / Q000378:metabolism*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D012709:Serum Albumin; D006493:Heparin; D014357:Trypsin; D005947:Glucose