Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Oxygen transfer characteristics of the blood of reedfish, Erpetoichthys calabaricus

Oxygen transport characteristics and phosphate compounds were measured in the blood of reedfish, Erpetoichthys calabaricus, a bimodal breather. Blood from reedfish possessed the following values (mean +/- SD): hematocrit (21.7 +/- 0.4%), hemoglobin concentration (7.53 +/- 1.75 g%), red blood cell count (0.45 +/- 0.10 X 10(6)/mm3) and oxygen capacity (10.1 +/- 2.3 vol%). Although hematocrit, hemoglobin concentration, red blood cell count and oxygen capacity were all highly intercorrelated (P less than 0.01 in all cases), none of these parameters were significantly correlated with sex, weight or length in our sixteen fish sample. Erythrocyte volumes equalled 480 micrometers3, showed less variation (CV = 10.4%) and did not correlate with any other measured variable. Blood oxygen dissociation curves were sigmoidal and the P50's equalled 17.34 +/- 3.04 at 1% CO2 and 25 degrees C. Mean Bohr shift (delta log P50/delta pH) was -0.274 +/- 0.087. Temperature strongly influenced blood oxygen affinity. At 1% CO2, delta log P50/delta T equalled 0.026 +/- 0.006 (mean +/- SD). These hematological properties indicate that the blood of reedfish is similar to those of other tropical air-breathering species. Concentrations of total phosphate in the erythrocytes and percentage of total phosphate bound as nucleotide triphosphates were high. Surprisingly, 2,3diphosphoglycerate was found which has been reported in the erythrocytes of only two other fish species. Blood characteristics of reedfish exposed to air for 4 hr with one exception (Hill numbers) were not significantly different from water exposed controls. This suggests that the reedfish does not possess blood respiratory mechanisms to facilitate respiration solely by air-breathing.     

Ventilatory and metabolic responses of burrowing owls, Athene cunicularia, to moderate and extreme hypoxia: analysis of the hypoxic ventilatory threshold vs. hemoglobin oxygen affinity relationship in birds

We measured ventilation, oxygen consumption and blood gases in burrowing owls (Athene cunicularia) breathing moderate and extreme hypoxic gas mixtures to determine their hypoxic ventilatory threshold (HVT) and to assess if they, like other birds and mammals, exhibit a relationship between HVT and hemoglobin O2 affinity (P(50)) of their blood. An earlier report of an attenuated ventilatory responsiveness of this species to hypoxia was enigmatic given the low O2 affinity (high P(50)) of burrowing owl hemoglobin. In the current study, burrowing owls breathing 11% and 9% O2 showed a significantly elevated total ventilation. The arterial partial pressure of oxygen (PaO2) at which ventilation is elevated above normoxic values in burrowing owls was 58 mm Hg. This threshold value conforms well to expectations based on the high P(50) of their hemoglobin and the HVT vs. P(50) relationship for birds developed in this study. Correcting for phylogenetic relatedness in the multi-species analysis had no effect on the HVT vs. P(50) relationship. Also, because burrowing owls in this study did not show a hypometabolic response at any level of hypoxia (even at 9% O2); HVT described in terms of percent change in oxygen convection requirement is identical to that based on ventilation alone.     

Ventilatory and metabolic responses of burrowing owls, Athene cunicularia, to moderate and extreme hypoxia: analysis of the hypoxic ventilatory threshold vs. hemoglobin oxygen affinity relationship in birds.

We measured ventilation, oxygen consumption and blood gases in burrowing owls (Athene cunicularia) breathing moderate and extreme hypoxic gas mixtures to determine their hypoxic ventilatory threshold (HVT) and to assess if they, like other birds and mammals, exhibit a relationship between HVT and hemoglobin O2 affinity (P(50)) of their blood. An earlier report of an attenuated ventilatory responsiveness of this species to hypoxia was enigmatic given the low O2 affinity (high P(50)) of burrowing owl hemoglobin. In the current study, burrowing owls breathing 11% and 9% O2 showed a significantly elevated total ventilation. The arterial partial pressure of oxygen (PaO2) at which ventilation is elevated above normoxic values in burrowing owls was 58 mm Hg. This threshold value conforms well to expectations based on the high P(50) of their hemoglobin and the HVT vs. P(50) relationship for birds developed in this study. Correcting for phylogenetic relatedness in the multi-species analysis had no effect on the HVT vs. P(50) relationship. Also, because burrowing owls in this study did not show a hypometabolic response at any level of hypoxia (even at 9% O2); HVT described in terms of percent change in oxygen convection requirement is identical to that based on ventilation alone.     

The oxygen uptake of Sarotherodon niloticus L. and the oxygen binding properties of its blood and hemolysate (Pisces: Cichlidae)

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.     

Acidosis attenuates P2X purinergic vasoconstriction in skeletal muscle arteries

Vasoconstriction via alpha(2)-receptors is known to be sensitive to acidic pH, but little is known about the pH sensitivity of P2X receptors. ATP is a cotransmitter released with norepinephrine from the sympathetic nerves and causes vasoconstriction via P2X purinergic receptors on vascular smooth muscle. We hypothesized that reductions in pH would attenuate P2X-mediated vasoconstriction in iliofemoral artery rings. Twenty-five rats were killed, and the iliac and femoral arteries were dissected out and placed in modified Krebs-Henseleit buffer. The arteries were cut into 2-mm sections and mounted in an organ tissue bath. Tension (g) was measured during a potassium chloride and norepinephrine challenge (maximal tension). The arteries were then exposed to alpha,beta-methylene ATP (10(-7)-10(-3) M; n = 13) or phenylephrine (10(-7)-10(-4) M; n = 6) with a tissue bath pH of 7.8, 7.4, and 7.0. Dose-response curves were fit with nonlinear regression analysis to calculate the EC(50) and slope. The peak tension with alpha,beta-methylene ATP was lower during pH 7.0 (1.37 +/- 0.09 g) compared with pH 7.8 (1.90 +/- 0.12 g). EC(50) was highest with pH 7.4 (-5.38 +/- 0.18 log M alpha,beta-methylene ATP) and lowest with pH 7.0 (-4.9 +/- 0.10 log M alpha,beta-methylene ATP). The slopes of the dose-response curves were not different. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) abolished contraction caused by the addition of alpha,beta-methylene ATP (n = 6). There was no effect of pH on phenylephrine dose-response curves. These data indicate that the vasoconstrictor response to alpha,beta-methylene ATP is sensitive to pH and that lower pH attenuates the response of P2X purinergic receptors.     

Blood acid-base status, whole blood and whole body buffer values in sand rats (Psammomys obesus) exposed to hypercapnia

Arterial blood acid-base status of unanesthetized sand rats (Psammomys obesus) were studied under normocapnic and hypercapnic conditions, and compared to those obtained for the albino rat (Rattus norvegicus). The average control blood pH: 7.396 +/- 0.034; PaCO2: 30.5 +/- 2.9 mmHg; HCO-3: 18.8 +/- 2.5 mM/l; and HCO-3 std: 20.9 +/- 2.1 (N = 15) obtained here for the sand rat are in the lower range of values found in other mammals and indicate a status of partially compensated metabolic acidosis. The blood buffer values of the sand rat, delta log PCO2/delta pH = -2.32 +/- 0.35 (N = 25) are significantly higher than those found here for the rat, delta log PCO2/delta pH = -1.51 +/- 0.10 (N = 39), and those reported for other mammals. This high blood buffer value may be related to the natural high mineral diet of the sand rat. The in vivo (whole body) buffer value delta log PaCO2/delta pH = -1.41 and -1.65 for the sand rat and the rat found here are higher than those reported for the man and dog and may represent a physiological adaptation to the hypercapnic conditions prevailing in underground burrows.     

Non-cooperative oxygen binding in the erythrocytes of pre-implanted sheep embryos

The whole-blood oxygen equilibrium curve in sheep embryos at 17 days gestation was essentially hyperbolic, indicating non-cooperative O2 binding with Hill's coefficient, n approximately equal to 1.2. O2 affinity was higher in embryonic blood (P50 = 7.1 mmHg at pH = 7.4 and 39 degrees C) than in maternal blood (P50 = 32.6 mmHg at pH = 7.4 and 39 degrees C). The Bohr effect was apparently smaller in the embryo (delta log P50/delta pH = --0.52) than in maternal blood (--0.36).     

Hemoglobin and oxygen: different affinities in two species of rodents (Mus musculus and Pitymys duodecimcostatus)

Five different hemoglobins have been demonstrated by polyacrylamide-gel disk electrophoresis in the species Mus musculus. Oxygen affinities of hemoglobin (P50) from Mus musculus and Pitymys duodecimcostatus hemolysates were determined at pH 7.4 and 37 degrees C. Values obtained for delta log P50/delta pH in hemolysates from both species point out a more pronounced Bohr effect in Pitymys duodecimcostatus.     

Reciprocal translocations in germ cells of male mice receiving external gamma-irradiation

Data reported in the literature up to 1985 on reciprocal translocation induction in male mouse germ cells by external gamma-ray doses ranging from 0.5 to 6.0 Gy delivered at fixed dose rates were analyzed. On the assumption of a non-threshold linear dose response, zero effect at zero dose, and a center of distribution lying on an approximately straight line, calculations were made of linear regression coefficients. These coefficients (b), as a function of the dose rate (P), were well fitted by two straight lines: b = (3.15 +/- 0.59 log P) X 10(-6) for dose rates from 0.01 to 0.1 mGy/min; and b = (7.52 +/- 3.86 log P) X 10(-6) for dose rates ranging from 0.06 to 1.2 X 10(3) mGy/min. The intersection point of these two lines determined the so-called threshold level of the dose rate, namely, 4.6 X 10(-2) mGy/min, at which the effectiveness of external gamma-irradiation is not expected to exceed 2.36 X 10(-6)/mGy. In addition, experiments were undertaken in which yields were recorded of reciprocal translocations in germ cells of male mice exposed to 0.9 Gy of gamma-radiation at dose rates ranging from 6.14 X 10(-3) to 6.14 X 10(2) mGy/min (6 levels); comparisons were made with data published up to 1985 from similar studies using other fixed doses. To do this, translocation yields were expressed as relative yields (F) and their relationship to the dose rate (P) for the individual fixed doses was represented by an equation of the type: F = alpha + beta log P. For most of the equations, the regression coefficients were in good agreement and a single relationship was obtained to represent them. From the analysis performed it follows that, within the 0.6-6.0 Gy dose range, the pattern of the F vs. P relationship is unaffected by the dose. This supports the initial assumption that for the dose range up to 6.0 Gy the dose response for the reciprocal translocation yield is a non-threshold straight-line relationship.     

Arctic life adaptation--III. The function of whale (Balaenoptera acutorostrata) hemoglobin

1. The oxygen binding properties of the hemoglobin from the Lesser Rorqual, Balaenoptera acutorostrata, has been investigated with respect to the possible effects of organic phosphates on gas transport in arctic environments. 2. The intrinsic oxygen affinity of the hemoglobin is high and strongly modulated by the effects of organic phosphates. 3. In the absence of organic phosphates, the temperature sensitivity of oxygen binding expressed by the heat of oxygenation, delta H, is -16.2 kcal/mol when corrected for the heat of oxygen in solution. 4. In the presence of organic phosphates there is a marked decrease in the temperature sensitivity delta H approximately -5 kcal/mol). 5. This feature is of great importance for oxygen unloading in the flippers and the tail, where the temperature is lower than the trunk of the whale. 6. Furthermore the organic phosphates strongly increase the Bohr coefficient, delta log P50/delta pH, from less than -0.3 in stripped hemoglobin to about -1.5 when the hemoglobin is saturated with P6-inositol. 7. This feature may be of great physiological importance by reducing the CO2 tension and acidosis after a prolonged dive.     

Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells

By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH. During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP. Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed [Rosen, B. P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.     

Adaptations to hypercapnic conditions in the nutria (Myocastor coypus)--in vivo and in vitro CO2 titration curves

Arterial blood acid-base status of unanesthetized, unrestrained nutria was studied during exposure to 5, 10 and 14.5% CO2 for 6 hr. Control values, pH = 7.426 +/- 0.037, PaCo2 = 36.5 +/- 3.1 mmHg and [HCO-3] = 24.3 +/- 2.5 mM/1 (n = 24), are within the normal range reported for other mammals. Values after 6 hr of exposure to 10% CO2 were: pH = 7.355 +/- 0.043, PaCO2 = 71.0 +/- 3.6 mmHg and [HCO-3] = 38.0 +/- 4.1 mM/l (n = 5). Arterial blood buffer slopes, obtained from the in vitro titration curve, did not show any pattern of adaptation to hypercapnia. Whole body buffer slopes, calculated from the in vivo CO2 titration curve, showed significantly higher values for the nutria than for the rat, dog and man, under comparable conditions [beta(delta HCO-3/delta pH)] = 57.0 slykes for nutria, 32.6 for rat and 11.8 for man. delta H+/delta PaCO2 = 0.38. mM/l/mmHg for nutria, 0.55 for rat and 0.76 for man. The results suggest that the nutria possesses an efficient metabolic mechanism for regulation of pH level during exposure to hypercapnic conditions.     

17O and 14N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by 17O and 14N n.m.r. spectroscopy. The 17O and 14N transverse relaxation time (T2p) and chemical shift (delta omega a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 +/- 0.02, and pH = 6.45 +/- 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary 17O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift delta omega a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (-1.6 +/- 0.1) X 10(5) Hz and the entropy and enthalpy (32.0 +/- 3.0) kJ/mol and (-12.0 +/- 1.0) e.u, respectively. Information obtained from 17O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H2O)6(2+), CoL X 5H2O and CoL2 X 4H2O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure dependence of equilibria and kinetics of Escherichia coli ribosomal subunit association

Equilibria and rates were observed over the ranges 1-1600 atm, 3-10 mM Mg2+, at 60 mM NH4Cl, pH 7.5, 20 degrees C, by light scattering. The main reaction is accurately represented at all conditions by the following phenomenological equations. 30 S + 50 S = 70 S, KA70 = ka/kd = [70 S]/[30 S][50 S] The equilibrium constants obey simple rules: the volume of association, delta VA0, has the constant value 242 +/- 9 ml/mol, independent of pressure, at all Mg2+ concentrations; the derived values of log KA70 at 1 atm increase linearly with log [Mg2+] at a slope of 7.5. In contrast, the rate constants show a clear break at 6 mM Mg2+: below 6 mM, log ka decreases with pressure with a delta Va of 105 +/- 9 ml/mol and increases with log [Mg2+] at a slope of 4.9; above 6 mM, these values are halved; a split can actually be seen at 6 mM Mg2+, near 500 atm. The usual two-step mechanism for second order reactions in solution, which would insert a 70 S' species, either an encounter complex or a true low concentration steady state intermediate, into the above equation can accommodate these results: as [Mg2+] increases, the rate of transformation of 70 S' into 70 S finally predominates over the rate of dissociation of 70 S' into subunits. The bulk of the pressure effects and all of the [Mg2+] dependence arise from the progressive increase in delta GA0 (electrostatic) that occurs when 30, 50, and 70 S particles all lose equivalent fractions of their internal Mg2+ in response to increases in pressure or decreases in [Mg2+].     

Life cycle and biology of Eimeria lettyae sp. n. (Protozoa: Eimeriidae) from the northern bobwhite, Colinus virginianus (L.)

A new species of coccidium, Eimeria lettyae sp. n. (Protozoa: Eimeriidae) was recovered from feces of the northern bobwhite, Colinus virginianus (L.), from Pennsylvania and Florida. Oocysts measured 21.1 microns (16.4 to 25.8) by 17.2 microns (14.1 to 21.2); index (L/W ratio) = 1.22. Oocysts lacked a micropyle, residuum, and polar granules. Sporozoites penetrated the upper 1/2 of the villi, then moved to the lamina propria at the base of the villi. There were five asexual generations, all of which developed above the nucleus of the host cell. Meronts measured 9.4 X 7.0 microns, 18.6 X 11.2 microns, 11.8 X 10.1 microns, 7.1 X 6.2 microns, and 20.2 X 12.8 microns, respectively. These matured at 32, 40, 48, 56, and 72 hr postinoculation (PI) and contained 12, 50+, 24 to 36, 12 to 24, and 50+ merozoites, respectively. Infection was most intense in the duodenum although some gamonts were found in the ileum and ceca. The prepatent period was 88 to 91 hr PI. Sporulation time was 18 hr at 25 C. The peak of oocyst production was broad and extended from 4 days PI through 14 days PI. Oocysts were passed for at least 67 to 76 days PI. Eimeria lettyae sp. n. did not infect chickens (Gallus domesticus), domestic turkeys (Meleagris gallopavo), ring-necked pheasants (Phasianus cochicus), chukar partridge (Alectoris graeca), or Japanese quail (Coturnix coturnix). Immunizing bobwhite with E. lettyae sp. n. did not protect against challenge with E. dispersa. Immunizing bobwhites 25 times with 10(2) or 10(3) sporulated oocysts of E. lettyae did not entirely eliminate oocyst production following challenge with the same species.     

Acid-base and respiratory properties of a buffered bovine erythrocyte perfusion medium

Current research in organ physiology often utilizes in situ or isolated perfused tissues. We have characterized a perfusion medium associated with excellent performance characteristics in perfused mammalian skeletal muscle. The perfusion medium consisting of Krebs-Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its gas-carrying relationships and its response to manipulation of acid-base state. Equilibration of the perfusion medium at base excess of -10, -5, 0, 5, and 10 mmol X L-1 to humidified gas mixtures varying in their CO2 and O2 content was followed by measurements of perfusate hematocrit, hemoglobin concentration, pH, Pco2, Cco2, Po2, and percent oxygen saturation. The oxygen dissociation curve was similar to that of mammalian bloods, having a P50 of 32 Torr (1 Torr = 133.3 Pa), Hill's constant n of 2.87 +/- 0.15, and a Bohr factor of -0.47, showing the typical Bohr shifts with respect to CO2 and pH. The oxygen capacity was calculated to be 190 mL X L-1 blood. The carbon dioxide dissociation curve was also similar to that of mammalian blood. The in vitro nonbicarbonate buffer capacity (delta [HCO3-] X delta pH-1) at zero base excess was -24.6 and -29.9 mmol X L-1 X pH-1 for the perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were quantified. The data were used to construct an acid-base alignment diagram for the medium, which may be used to quantify the flux of nonvolatile acid or base added to the venous effluent during tissue perfusions.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI Kunitz inhibitor) to human Lys77-plasmin has been investigated. Ka values decrease with decreasing pH, reflecting the acid-pK and -midpoint shifts, upon BPTI binding, of a single ionizable group, between pH 5 and 9, and of a three-proton transition, between pH 3 and 5. At pH 8.0, values of thermodynamic parameters for BPTI binding to human Lys77-plasmin are: Ka = 1.2 X 10(9) M-1, delta G degree = -12.2 kcal/mol, and delta S degree = +49 entropy units (at 21 degrees C); and delta H degree = +2.3 kcal/mol (temperature independent between 5 degrees C and 45 degrees C; 1 kcal = 4184 J). BPTI binding properties of human Lys77-plasmin have been analysed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates. Considering the known molecular structures of homologous serine (pro)enzymes, or Kunitz and Kazal-type inhibitors and of their complexes, the observed binding behaviour of BPTI to human Lys77-plasmin was related to the inferred stereochemistry of the enzyme-inhibitor contact region.     

Temperature regulation in adult quail (Colinus virginianus) during acute thermal stress

1. Evaporative heat loss, O2 consumption, CO2 production, and internal body temperature were measured in unanesthetized, unrestrained bobwhite (Colinus virginianus) at specific ambient temperatures (Ta). 2. No significant change in body temperature occurred at any Ta tested, but metabolic heat production (H) increased from 42.17 W/m2 at Ta 35 degrees C to 102.89 W/m2 at Ta 10 degrees C. 3. Evaporative heat loss (E) increased approximately two-fold from Ta 10-35 degrees C, with E/H increasing exponentially over the same temperature range. 4. No significant change in thermal insulation occurred from Ta 10-30 degrees C. 5. Combined convective and radiative heat transfer for the bobwhite was 2.96 W/m2 X C from Ta 10-35 degrees C.