The lipocalin alpha1-microglobulin has radical scavenging activity.
The lipocalin alpha(1)-microglobulin (alpha(1)m) is a 26-kDa glycoprotein present in plasma and in interstitial fluids of all tissues. The protein was recently shown to have reductase properties, reducing heme-proteins and other substrates, and was also reported to be involved in binding and scavenging of heme and tryptophan metabolites. To investigate its possible role as a reductant of organic radicals, we have studied the interaction of alpha(1)m with the synthetic radical, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS radical). The lipocalin readily reacted with the ABTS radical forming reduced ABTS. The apparent rate constant for this reaction was 6.3 +/- 2.5 x 10(3) M(-1) s(-1). A second reaction product with an intense purple color and an absorbance maximum at 550 nm was formed at a similar rate. This was shown by liquid chromatography/mass spectrometry to be derived from covalent attachment of a portion of ABTS radical to tyrosine residues on alpha(1)m. The relative yields of reduced ABTS and the purple ABTS derivative bound to alpha(1)m were approximately 2:1. Both reactions were dependent on the thiolate group of the cysteine residue in position 34 of the alpha(1)m polypeptide. Our results indicate that alpha(1)m is involved in a sequential reduction of ABTS radicals followed by trapping of these radicals by covalent attachment. In combination with the reported physiological properties of the protein, our results suggest that alpha(1)m may be a radical reductant and scavenger in vivo.
Akerström
Bo
B
Department of Clinical Sciences, Lund University, 22184 Lund, Sweden. bo.akerstrom@med.lu.se
Maghzal
Ghassan J
GJ
Winterbourn
Christine C
CC
Kettle
Anthony J
AJ
D000478:Alkylation; D000510:Alpha-Globulins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D052160:Benzothiazoles / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics; D016166:Free Radical Scavengers / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D054834:Lipocalins / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012463:Saliva / Q000737:chemistry; D012694:Serine / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry / Q000378:metabolism*; D013666:Tears / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D000510:Alpha-Globulins; D052160:Benzothiazoles; D016166:Free Radical Scavengers; D007202:Indicators and Reagents; D054834:Lipocalins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C016001:alpha-1-microglobulin; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D012694:Serine; D006639:Histidine; D014357:Trypsin