Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: a MALDI-TOF-MS study.
In this work, MALDI-TOF-MS analysis was performed to obtain information on the molecular mass of the different subunits from the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy-form. Experiments were performed for the whole protein at pH 7.0, for the partially dissociated protein at pH 9.0, and for the fraction obtained from gel filtration in Sephadex G-200, at pH 9.0, corresponding to the isolated monomer d. Besides that, experiments were performed for the whole protein treated with 2-mercaptoethanol in order to monitor the effects of reduction of the disulfide bonds, which are expected to maintain the trimer (abc) in the native molecule. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16,355+/-25 and 16,428+/-24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c on the basis of comparison with Lumbricus terrestris hemoglobin literature data are observed. Their molecular masses were 18,258+/-30, 16,492+/-24 and 17,363+/-17 Da, respectively. Two linker chains for HbGp were also observed at 25,817+/-50 and 26,761+/-16 Da, and this result is different from HbLt, where four linker chains were reported in the range 24-32 kDa. Finally, trimers (abc) were observed at 51-52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant extracellular hemoglobin.
Oliveira
Marilene Silva
MS
Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, USP, C.P. 780, 13560-970 São Carlos, SP, Brazil.
Moreira
Leonardo Marmo
LM
Tabak
Marcel
M
D000818:Animals; D000845:Annelida / Q000737:chemistry*; D004220:Disulfides; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010108:Oxyhemoglobins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D006454:Hemoglobins; D010108:Oxyhemoglobins