This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The extracellular hemoglobin of annelids and tube worms are multisubunit proteins of up to 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having O2 binding chains and """"""""linker"""""""" chains. The linker chain L1 of the hemoglobin of Lumbricus terrestris contains a 38-39 residue segment with a repeating pattern of cysteinyl residues (CysX6)3 -CysX5-CysX10-Cys. This pattern, not present in any globin sequence, corresponds exactly tao the cysteine rich repeats of the ligand binding domain of the low density lipoprotein (LDL) receptor of man and Xenopus laevis. The disulfide connectivity of these domains has not been determined in the LDL receptor. The determination of the disulfide bonds in the linker chains is therefore an important first step in the understanding of the interaction between apolipoprotein E and the LDL receptor. Linker chain L1 was digested with a number of enzymes and the resulting peptide mixtures were a nalysed by matrix-assisted laser desorption mass spectrometry. Due to the huge number of possible disulfide bridges, the assignment of peaks to disulfide bridges containing peptides was extremely difficult. Therefore HPLC fractionation of peptide mixtures was carried out. For the identification of disulfide containing peptides fractions of interest further enzymatic and chemical reactions are performed. Identification of the resulting peptide products provides information that is useful for mapping the disulfide linkages. Ultimately, we found that MALDI-ITMS of the enzyme digested protein provided the highest quality mapping data. A paper describing the technique and discussing the results has been published. we are currently gearing up to carefully define the carbohydrate structures in the earthworm Hb chains.
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