Electrospray mass spectrometric investigation of the binding of cis-parinaric acid to bovine beta-lactoglobulin and study of the ligand-binding site of the protein using limited proteolysis.
The binding property of parinaric acid, a polyunsaturated fatty acid, to bovine beta-lactoglobulin, has been studied by electrospray ionization mass spectrometry. Stable complexation was observed under acidic conditions in a molar ratio of 1:1. Competitive complexation experiments were performed using saturated and unsaturated fatty acid standards with different chain lengths and number of double bonds to study the specificity of the interaction. It can be concluded that formation of the parinaric acid-lactoglobulin complex is preferred even if the molar concentration of the other fatty acids is ten times higher. In cases of specific complex formation the protein must have an active site that is a good acceptor for the ligand molecule. Limited trypsinolysis was performed on the lactoglobulin molecule to identify which part is responsible for the complexation. An intermediate tryptic fragment with molecular mass of 5200 Da was found to have the same ability to bind parinaric acid as the intact protein. This disulfide-bonded residue, [41-70]S-S[149-162], might thus be involved in the specific complexation of parinaric acid to beta-lactoglobulin. This conclusion is consistent with previous information on this binding site.
Imre
Tímea
T
Department of Mass Spectrometry, Institute of Chemistry, Chemical Research Center, PO Box 17, 1525 Budapest, Hungary.
Zsila
Ferenc
F
Szabó
Pál T
PT
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005227:Fatty Acids / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008970:Molecular Weight; D019308:Palmitic Acid / Q000737:chemistry; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism
D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D008024:Ligands; D019308:Palmitic Acid; C013641:parinaric acid