Fluorometric and mass spectrometric analysis of nonenzymatic glycosylated albumin.
Albumin is the major transport protein in blood and intramolecular movement contributes to this function. Nonenzymatic glycosylation (NEG) of albumin occurs in diabetes and, in this study, fluorometric methods were used to determine the effect of increasing levels of NEG upon intramolecular movement in human serum albumin. Low levels of NEG significantly reduced and left-shifted Trp fluorescence, reduced quenching by acrylamide and inhibited penetration of bis-ANS, while these changes became only modestly more pronounced at higher levels of NEG. Mass spectrometry of tryptic and CNBr NEG-HSA fragments identified potential glycosylation sites and demonstrated only late glycosylation of the C- and N-terminal regions of the protein. Similar changes in diabetes may contribute to altered transport function in these patients.
Zoellner
H
H
Department of Oral Pathology and Oral Medicine, University of Sydney, Westmead Hospital Dental Clinical School, New South Wales, Australia. hansz@dental.wsahs.nsw.gov.au
Hou
J Y
JY
Hochgrebe
T
T
Poljak
A
A
Duncan
M W
MW
Golding
J
J
Henderson
T
T
Lynch
G
G
D020106:Acrylamide / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001665:Binding Sites / Q000502:physiology; D003488:Cyanogen Bromide / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry; D005947:Glucose / Q000737:chemistry*; D017127:Glycation End Products, Advanced / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D012709:Serum Albumin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D010446:Peptide Fragments; D012709:Serum Albumin; D020106:Acrylamide; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan; D014357:Trypsin; D005947:Glucose; D003488:Cyanogen Bromide