ELSEVIER Biochimica et Biophysica Acta 1249 (1995) 109-115 BB Biochific~a et Biophysica /Eta The hemoglobin system of the hagfish Myxine glutinosa: aggregation state and functional properties Angela Fago, Roy E. Weber * Departme~t of Zoophysiology, Institute of Biological Sciences, University of Aarhus, DK 8000 Aarhus C, Denmark Received 24 October 1994; revised 7 February 1995; accepted 15 February 1995 Abstract Hemoglobin (Hb) from the hagfish Myxine glutinosa is composed of six major monomeric subunits. Some of these subunits aggregate to dimers at low pH, and to tetramers when deoxygenated and at high protein concentration. The aggregation is inhibited by the presence of KC1. Oxygen equilibrium studies show the presence of a small Bohr effect which is strongly reduced by KC1, indicating that it originates from pH-dependent aggregation. ATP and DPG cause a similar decrease in the Bohr effect. 02 affinity is dependent on protein concentration, temperature and presence of CO 2. Cooperativity is practically absent. 02 binding properties of the separated aggregating and non-aggregating Hbs purified at low pH cannot account for the functional properties of the composite hemolysate, suggesting the presence of other subunits interactions. The results are discussed in relation to literature data for other cyclostome Hbs and for M. glutinosa Hb, where the presence of three major monomeric Hbs and a possible CO2-dependent aggregation had been reported. Keywords: Hemoglobin; Aggregation; Oxygen binding; Chloride effect; CO2 effect; Bohr effect; Cyclostome; (Hagfish); (M. glutinosa) I. Introduction Hagfishes like Myxine glutinosa and the more recent lampreys are the most primitive living craniate vertebrates belonging to the class Agnatha (jawless fish) that have existed since the Ordovician period, more than 400 million years ago (cf. [1,2]). "[he large differences between the primary structures of hagfish and lamprey Hbs are consis- tent with evolutionary divergence between the two families already 400-500 million years ago, i.e., well before the ancestral separation between myoglobin and hemoglobin chains [3]. Moreover, while lamprey Hb could be consid- ered closer to a-type chains of higher vertebrates [4,5] hagfish Hbs cannot be classified as a- or /3-type globins, as indicated by the amino-acid sequence of one Hb compo- nent from M. glutinosa [3]. Knowledge of the structural and functional propertie,; of hagfish Hbs thus is pivotal for understanding the evolution of vertebrate Hbs. Despite numerous studies on structural and functional properties of lampreys (cf. [4-21]), httle remains known about Hbs of the more primitive hagfishes. * Corresponding author. E-mail: rw@bio.aau.dk. Fax: +45 86 194186. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10167-4838(95)00031-3 Lamprey Hbs are monomeric when oxygenated but aggregate to form dimers or tetramers upon deoxygenation. Cooperativity of 02 binding (which is saturation-depen- dent) and the large Bohr effect of these Hbs are the result of ligand-induced dissociation of low 02 affinity aggre- gates into high affinity monomers (e.g., [8,15,17,22]). The Hbs from the hagfishes Epatretus cirrhatus [23] and E. burgeri [24] show a tendency to aggregate when deoxy- genated especially at low pH and high concentration, but their cooperativity and Bohr effects are much smaller than those found in lampreys Hbs. Hagfish hemolysates are known to show multiplicity and polymorphism of Hbs components [24,25]. M. glutinosa hemolysate was re- solved into three major Hbs by isoelectric focusing [26]. These Hbs showed apparent molecular masses ranging from 20 500 to 28 000 daltons [27], which are higher than the values for monomeric Hbs. Bauer et al. [2] attribute increases in the Bohr effect and cooperativity of 0 2 bind- ing of the hemolysate in the presence of CO 2 to aggrega- tion of the Hb components. These findings call for an investigation of the relationships between functional and aggregational properties in the hagfish Hb system, under varying physico-chemical conditions, which describes the aim of this paper.