Purification and properties of a small lipid-binding protein from the hemolymph of Triatoma infestans.
A small lipid-binding protein (sLBP) was purified from the hemolymph of the blood-sucking bug Triatoma infestans. Its isolation involved size exclusion-high performance liquid chromatography (HPLC) followed by anion exchange chromatography-HPLC. The molecular weight of the protein, as determined by gel permeation chromatography, was 20 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved the protein into a single polypeptide with M(r) approximately equal to 16 kDa. The sLBP contains 6% lipids. Diacylglycerols represent the major lipid class, whereas phosphatidyl-choline, phosphatidyl-ethanolamine, free fatty acids and hydrocarbons were found in minor amounts. The amino acid composition indicated a high content of aspartic and glutamic acids and non-polar aliphatic amino acids. The N-terminal sequence did not resemble the sequence of any other previously reported insect hemolymph protein. Far-UV circular dichroism suggested that sLBP adopts a conformation rich in beta-sheet structure. The presence of this protein in hemolymph, fat body and unfertilized eggs was explored throughout the last nymphal and adult stages of the insect by Western blot assays. These assays indicated that sLBP is particularly abundant in hemolymph. A high concentration of sLBP was also detected in the fat body of the nymphs.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006458:Hemolymph / Q000378:metabolism*; D015151:Immunoblotting; D019476:Insect Proteins / Q000737:chemistry; D007313:Insecta / Q000737:chemistry*; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008297:Male; D013050:Spectrometry, Fluorescence; D014018:Tissue Distribution; D014461:Ultracentrifugation