Vertebrate hemoglobin, contained in erythrocytes, is a globular protein with a quaternary structure composed of 4 globin chains (2 alpha and 2 beta) and a prosthetic group named heme bound to each one. Having myoglobin as an ancestor, hemoglobin acquired the capacity to respond to chemical stimuli that modulate its function according to tissue requirements for oxygen. Fish are generally submitted to spatial and temporal O2 variations and have developed anatomical, physiological and biochemical strategies to adapt to the changing environmental gas availability. Structurally, most fish hemoglobins are tetrameric; however, those from some species such as lamprey and hagfish dissociate, being monomeric when oxygenated and oligomeric when deoxygenated. Fish blood frequently possesses several hemoglobins; the primary origin of this finding lies in the polymorphism that occurs in the globin loci, an aspect that may occasionally confer advantages to its carriers or even be a harmless evolutionary remnant. On the other hand, the functional properties exhibit different behaviors, ranging from a total absence of responses to allosteric regulation to drastic ones, such as the Root effect.

This work reports endoglucanase and beta-glucosidase production by the thermophilic fungusMyceliophthora heterothallicain solid-state (SSC) and submerged (SmC) cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. Forβ-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSCβ-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl₃exerted the strongest inhibition for endoglucanases andβ-glucosidases.

We have previously proposed a role of hydration in the allosteric control of hemoglobin based on the effect of varying concentrations of polyols and polyethers on the human hemoglobin oxygen affinity and on the solution water activity (Colombo, M. F., Rau, D. C., and Parsegian, V. A. (1992) Science 256, 655-659). Here, the original analyses are extended to test the possibility of concomitant solute and water allosteric binding and by introducing the bulk dielectric constant as a variable in our experiments. We present data which indicate that glycine and glucose influence HbA oxygen affinity to the same extent, despite the fact that glycine increases and glucose decreases the bulk dielectric constant of the solution. Furthermore, we derive an equation linking changes in oxygen affinity to changes in differential solute and water binding to test critically the possibility of neutral solute heterotropic binding. Applied to the data, these analyses support our original interpretation that neutral solutes act indirectly on the regulation of allosteric behavior of hemoglobin by varying the chemical potential of water in solution. This leads to a displacement of the equilibrium between Hb conformational states in proportion to their differential hydration.