The purpose of this research is to investigate the mechanism(s) of hemoglobin association/dissociation. Its regulation in vivo and in vitro and its effect on hemoglobin function. The mollusc, Anadara ovalis, has erythrocytes which contain dimeric (HbMi) and tetrameric (HbMa) hemoglobins both of which dissociate to monomers upon oxidation with K3Fe(CN)6 (Borgese et al., 1987). In contrast, the marine teleost, Lophius amercanus (goosefish), has a single tetrameric hemoglobin which forms aggregates ranging from 160.000 to greater than 900,000 daltons when similarly oxidized (Borgese, 1986). While many species of elasmobranchs have multiple hemoglobins, some of which polymerize by disulfide bond formation, there have been no reports of hemoglobin aggregation among marine teleosts.
Our specific aims are: 1) to determine the effects of disulfide reducing agents on the inhibition and reversal of the Hb aggregation (goosefish, ocean pout) and monomerization (Anadara ovalis). Hemoglobins will be isolated by DE-52 chromatography and molecular weights determined by gel filtration. 2) We will determine whether aggregation can occur in intacts cells by incubating cells with permeable oxidants such as NaN02 followed by electron microscopy of the packed cells. 3) The complete red cell organic phosphate profile will be determined by Ag 1x8 anion exchange chromatography; the effect of the major organic phosphates are hemoglobin function will be determined using the Hem-O-Scan oxygen dissociation analyzer. 4) We will determine whether cells with metabolically stimulated reductase activity have a greater capacity to inhibit aggregation. 5) We will determine the role of SH groups on thermal stability of goosefish and Anadara ovalis HbMa. 6) Urea gel electrophoresis and HPLC will be used to determine whether goosefish hemoglobin is a homo or heterotetramer. Amino acid composition of the isolated chains and the sequencing of these unusual hemoglobins (16 SH/tetramer-goosefish and monomer formation of Anadara HBs) are further steps in explaining struc- ture-function-property relationships. In addition to providing an explanation for apparent Hb polymorphism, alterations in the normal quaternary hemoglobin structure by polymerization or dissociation may have serious consequences if cell morphology and/or oxygen affinity are adversely affected.
