title: Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.
abstract: Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.
authors.affiliation: Upjohn Laboratories, Upjohn Company, Kalamazoo, Michigan 49001.
authors.forename: A W
authors.initials: AW
authors.lastname: Yem
authors.forename: D E
authors.initials: DE
authors.lastname: Epps
authors.forename: W R
authors.initials: WR
authors.lastname: Mathews
authors.forename: D M
authors.initials: DM
authors.lastname: Guido
authors.forename: K A
authors.initials: KA
authors.lastname: Richard
authors.forename: N D
authors.initials: ND
authors.lastname: Staite
authors.forename: M R
authors.initials: MR
authors.lastname: Deibel
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003545:Cysteine; D007375:Interleukin-1 / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008213:Lymphocyte Activation; D008239:Lysine; D051379:Mice; D008809:Mice, Inbred C3H; D008961:Models, Structural; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology
chemical_list: D007375:Interleukin-1; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; C038518:acrylodan; D015081:2-Naphthylamine; D008239:Lysine; D003545:Cysteine