Chemical modification and nuclear magnetic resonance studies on human plasminogen kringle 4. Assignment of tyrosine and histidine resonances to specific residues in the sequence.
Modification of kringle 4 with tetranitromethane leads to the selective nitration of tyrosine 40 but on prolonged incubation with reagent, reaction of tyrosine 49 is also observed. Nitration of tyrosines 40 and 49 had no influence on the lysine-Sepharose affinity of kringle 4, indicating that these residues are not important for the functional integrity of the ligand-binding site. Comparison of the NMR spectra of native kringle 4 with those of kringle 4 in which tyrosine 40 or tyrosines 40 and 49 are nitrated permitted the identification of the resonances of these residues. These NMR studies also showed that the chemical modifications caused little perturbation of the three-dimensional structure of the protein. Cross-linking of lysine 35 and tyrosine 40 with 1,3-difluoro-4,6-dinitrobenzene demonstrates that in the kringle-fold the reactive epsilon-amino and phenolic groups of these residues can approach each other to a distance of 0.5 nm. NMR spectra of this kringle 4 species also confirmed the assignment of the resonances to tyrosine 40. NMR spectra of a kringle 4 derivative in which the disulphide bridge between cysteines 1 and 79 has been broken by selective reduction and alkylation showed that the core structure of the kringle-fold and the lysine-binding site are unaltered by this modification. This observation is in agreement with earlier results which showed that the lysine-Sepharose affinity of kringle 4 is not affected by reduction and alkylation of this disulphide bridge. Comparison of the NMR spectra of native and disulphide-cleaved kringle 4 aided in the assignment of resonances to residues adjacent to the site of modification (tyrosine 2 and histidine 3) and permitted the tentative assignment of the resonances of tyrosines 9 and 73.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D006639:Histidine / Q000032:analysis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009584:Nitrogen; D010446:Peptide Fragments / Q000032:analysis*; D010958:Plasminogen / Q000032:analysis*; D013774:Tetranitromethane; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D014443:Tyrosine; D006639:Histidine; D010958:Plasminogen; D004139:Dinitrofluorobenzene; C002041:1,5-difluoro-2,4-dinitrobenzene; D013774:Tetranitromethane; D009584:Nitrogen