RAPID AND SIMULTANEOUS MEASUREMENT OF ANODIC AND CATHODIC HAEMOGLOBINS AND ATP AND GTP CONCENTRATIONS IN MINUTE QUANTITIES OF FISH BLOOD

Abstract

Oxygen transport to vertebrate tissues is dependent upon (a) the intrinsic O2-binding properties of haemoglobin (Hb) and (b) modulation by allosteric effectors, such as nucleoside triphosphates (NTP), that depress Hb O2-affinity in fish red cells. While some species, such as flatfish, primarily use adenosine triphosphate (ATP) to modulate O2-binding to Hb, others, such as eel and carp, also use guanosine triphosphate (GTP) (Kono and Hashimoto, 1977; Leray, 1982; Weber and Jensen, 1988). Unlike mammals, fish exhibit high degrees of molecular and functional Hb heterogeneity, which is manifested interspecifically but is also evident from polymorphism in different individuals of the same species (Weber, 1990). While some species such as carp have multiple Hbs that migrate anodally in normal electrophoresis and exhibit similar intrinsic oxygenation properties and sensitivities to NTP (Gillen and Riggs, 1972), others such as trout and eel have both anodic and cathodic components that exhibit different intrinsic O2-binding properties and different sensitivities to cofactors. Having higher O2 affinities and lower Bohr effects, the cathodic Hbs may be better adapted for O2 transport under hypoxic, hypercapnic or acidotic conditions than the anodic ones from the same species (Hashimoto et al. 1960; Binotti et al. 1971; Weber et al. 1975; Weber, 1990). Ambient hypoxia induces decreases in erythrocytic NTP (Wood and Johansen, 1972), which safeguards blood O2-loading in the gills. The concentrations of the major NTPs and anodic or cathodic Hbs are thus important indices of respiratory and adaptational status in fish. Erythrocytic ATP and GTP levels are generally assayed by thin layer or column chromatography or enzymatically, total Hb is measured by spectrophotometry, and the anodic and cathodic composition of the Hbs present by electrophoresis or isoelectric focusing. We here report a method for rapid and simultaneous assay of ATP and GTP, and of anodic and cathodic Hbs, in minute (approximately 3 µl) quantities of fish blood. We used trout [Oncorhynchus mykiss, 101±8 g (s.e.m.); N=9], eel (Anguilla anguilla, 114±17 g; N=9) and carp (Cyprinus carpio, 1407±104 g; N=9) supplied by local pisciculturists. The fish were kept for at least 1 week in 1 m3 glass fibre tanks with aerated running fresh water at 15 °C before experimentation. Blood samples were drawn from the caudal blood vessels into heparinised syringes.