Heparin-binding histidine and lysine residues of rat selenoprotein P.
Selenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [(14)C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80-95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVSDHIAVY. Two other peptides (residues 178-189 and 194-234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pK(a) of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH.
Division of Gastroenterology, Department of Medicine, Clinical Nutrition Research Unit, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
R J
RJ
Hondal
S
S
Ma
R M
RM
Caprioli
K E
KE
Hill
R F
RF
Burk
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000493:pharmacokinetics / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D051149:Selenoprotein P; D051140:Selenoproteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D006639:Histidine; D006493:Heparin; D008239:Lysine; D004047:Diethyl Pyrocarbonate