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1 October 1998 Molecular Stability and Function of Mouse Hemoglobins
Kou Uchida, Michael P. Reilly, Toshio Asakura
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The hemoglobin types of mouse strains can be distinguished according to patterns observed on cellulose acetate electrophoresis. The two common mouse hemoglobin patterns are single and diffuse. The differences in the patterns result from differences in the β-globin chains of the hemoglobin molecules. Mice with the single hemoglobin pattern have one β-globin type identified as β-single (Hbbs), whereas mice with the diffuse hemoglobin pattern have two different β-globin types identified as β-major (Hbbmaj) and β-minor (Hbbmin). We examined the functional and stability properties in these mouse hemoglobins, and the oxygen binding properties of red blood cells obtained from mice with four different hemoglobin phenotypes: Hbbs/Hbbs, Hbbs/Hbbmin, Hbbmin/Hbbmin and Hbbmaj/Hbbmin. The P50, the partial pressure of oxygen at which hemoglobin is half-saturated, of purified forms of Hbbs, Hbbmin and Hbbmaj was 14.8 ± 0.4 mm Hg, 13.3 ± 0.6 mm Hg and 13.6 ± 0.5 mm Hg, respectively. The n value, determined from the slope of the Hill plot was 2.45 to 2.59 for the mouse hemoglobins. The alkaline Bohr effects of purified HbbS, Hbbmin and Hbbmaj were 0.69, 0.61 and 0.60, respectively. The mechanical stability of Hbbs, Hbbmin and Hbbmaj, expressed by the first order kinetic constant, were 0.098 ± 0.01/min, 0.027 ± 0.013/min and 0.27/min, respectively. The P50 of red blood cell suspensions from lines of mice expressing Hbbs/Hbbs, Hbbmin/Hbbmin, Hbbs/Hbbmin and Hbbmaj/Hbbmin were 40.2 ± 1.8 mm Hg, 40.4 ± 1.5 mm Hg, 38.9 ± 1.4 mm Hg, and 38.7 ± 0.9 mm Hg, respectively.


Mice are characterized by polymorphisms of the hemoglobin β-chain (Hbb) (Whitney, 1978). Three β-chain haplotypes, single (Hbbs), diffuse and peculiar, have been described, both in wild and inbred strains (Russell and McFarland, 1974). In mice with a diffuse hemoglobin pattern, 80% of the adult β-globin is β-major (Hbbmaj), while the remaining 20% is β-minor (Hbbmin) (Russell and McFarland, 1974).

Mice are widely used in transgenic experiments and as animal models of various human diseases. Studies that involve expression of human hemoglobins in transgenic mice (Behringer et al., 1989; Ryan et al., 1990) require information on the structural and functional differences between human and mouse hemoglobins. Such studies on the structure-function relationship of mouse hemoglobins may provide important information regarding the regulatory mechanisms of hemoglobin oxygen affinity. In this paper, we measured the functional and stability properties of three different types of mouse hemoglobins, and the oxygen binding properties of red blood cells obtained from mice with four different hemoglobin haplotypes.



C57BL/6 and BALB/C mice used in this study were purchased from Jackson Labs (Bar Harbor, ME, USA) and Charles River (Wilmington, MA, USA), respectively. The hemoglobin phenotypes of C57BL/6 and BALB/C mice are Hbbs/Hbbs and Hbbmaj/Hbbmin, respectively. Mice heterozygous for a deletional form of β-thalassemia on a C57BL/6 background, also obtained from Jackson Labs, express both Hbbs and Hbbmin. The heterozygous mice were bred to homozygosity to obtain mice that produced only Hbbmin. The typing of mouse hemoglobin phenotypes was done by the method of Whitney (1978), using cellulose acetate electrophoresis with cystamine. On the basis of hemoglobin electrophoretic patterns, mouse RBC were characterized in four different hemoglobin phenotypes; homozygous Hbbs/Hbbs containing only Hbbs hemoglobin; heterozygous Hbbs/Hbbmin containing both Hbbs and Hbbmin hemoglobins; homozygous Hbbmin/Hbbmin containing only Hbbmin hemoglobin and Hbbmaj/Hbbmin containing both Hbbmaj and Hbbmin. Blood was drawn from the retro-orbital sinus into a heparinized microhematocrit capillary tube (Fisher Scientific, Pittsburgh, PA, USA). Normal human adult hemoglobin (HbA) and human sickle hemoglobin (HbS) were used as references for the mechanical and heat stability tests.

Purification of hemoglobin

Three different mouse hemoglobins, Hbbs, Hbbmaj and Hbbmin were examined in this study. Mouse globins were separated by reverse phase high pressure liquid chromatography (RP-HPLC), using a Dionex Series 4500i HPLC system (Sunnyvale, CA, USA) (Reilly et al., 1994). Approximately 25 μg hemoglobin was injected onto a Vydac C4 reversed-phase column (4.6 × 250 mm) (Hibernia, CA, USA) and eluted with a linear gradient of acetonitrile and 0.3% trifluroacetic acid as described by Shelton et al. (1984). RP-HPLC showed that hemolysates from homozygous Hbbs/Hbbs and homozygous Hbbmin/Hbbmin mice had single β-globin peaks corresponding to β-single and β-minor, respectively. Therefore, hemolysates of the Hbbs/Hbbs and Hbbmin/Hbbmin RBC were used for experiments without further purification (Fig. 1). To remove 2,3-DPG, the hemolysates were passed through a Sephadex-G-25 (Sigma Chemical Company, St. Louis, MO, USA) column. The Hbbmaj was purified from the blood of BALB/C mice (Fig. 1). After lysing the washed cells with a 20 mM Tris-5 mM EDTA solution (pH 7.2), the hemoglobin was dialyzed against a 10 mM phosphate buffer, pH 7.0, for 4 hr. The lysate was applied to a CMSephadex column (Sigma), and the hemoglobin was eluted from the column using a gradient of 10 mM phosphate buffer, pH 7.0, to 20 mM phosphate buffer, pH 8.0, as previously described (Adachi et al., 1980). Each chromatographic component was pooled and concentrated using a microconcentrator (Centricon-30, Amicon, Inc., Beverly, MA, USA).

Fig. 1

Oxygen dissociation curves of mouse hemoglobins. OEC for HbbS shows a right side shift compared to those of Hbbmin and Hbbmaj.


Oxygen equilibrium curves of the hemoglobins

Hemoglobin oxygen equilibrium curves (OEC), at a heme concentration of 100 mM, were determined in potassium phosphate buffer, pH 7.0, at 20°C with a Hemox-Analyzer (TCS, Southampton, PA, USA), an automatic apparatus that consists of a spectrophotometer cuvette fitted with a magnetic stirrer, gas exchange line and oxygen electrode. The gas electrode (oxygen tension; PO2) and spectrophotometer responses (% oxyhemoglobin; SO2) are recorded on the X and Y axes, respectively, of an X-Y recorder. The temperature of the cuvette is controlled by an external water bath circulating at a constant temperature (Festa and Asakura, 1979). Ten μl of 1% dimethylpolysiloxane (Union Carbide, NY, USA), an anti-foaming agent, and a stabilizing solution (0.001% hexamethyl-phosphoramide) were added to the solution. The methemoglobin reduction system (Hayashi et al., 1973) was used to prevent the oxidation of the hemoglobin. OEC were plotted according to the Hill equation with log (SO2/100-SO2) on the Y axes and log PO2 on the X axes. The slopes of the lines, Hill's n values, were determined in mouse hemoglobins. The alkaline Bohr effect was calculated from the pH dependence of the log P50 between pH 6.5 and 7.5. 2,3-DPG and inositol hexaphosphate (IHP) were products of Sigma.

Oxygen equilibrium curves of suspension of red cells

The OEC of red cells suspensions were determined by suspending the RBC in an isotonic TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer, pH 7.4 at 37°C. Ten μl of 1% dimethylpolysiloxane was added as an anti-foaming agent, and bovine serum albumin was added to yield a final concentration of 0.1% to prevent red cell hemolysis. The concentration of 2,3-DPG in the whole blood samples was measured using the method of Rose and Leibowitz (Rose and Liebowitz, 1970). Hematocrit (Ht) was measured following the centrifugation of the blood samples for 5 min in a Hawksley micro-hematocrit centrifuge.

Mechanical stability of hemoglobins

Mechanical shaking experiments were done as described previously (Asakura et al., 1974). Hemoglobin solutions from Hbbs/Hbbsand Hbbmin/Hbbmin mice and purified Hbbmaj were diluted in 0.1 M potassium phosphate buffer (pH 7.0) to a final heme concentration of 100 mM. Two mls of each solution were placed in a 10 × 50 glass vial and shaken with a Model 250 shaker (TCS, Southampton, PA, USA) at 60 Hz for various time intervals at room temperature. Following shaking, the vials were centrifuged at 15,000 × g, and the absorption spectrum of the hemoglobin remaining in the supernatant was recorded from 500 to 700 nm. The mechanical stability of the hemoglobins was expressed by the first order kinetic constant, K.

Heat denaturation experiments

Heat denaturation experiments were done using the oxy-forms of HbbS, Hbbmin, Hbbmaj, human HbA and human HbS. Each hemoglobin was diluted to yield a final heme concentration of 40 mM in 0.1 M phosphate buffer, pH 7.4, containing 5 mM EDTA. A continuous spectrum for each hemoglobin solution was recorded between 500 and 700 nm using a Hitachi U-3410 spectrophotometer (Hitachi, Tokyo, Japan). Hemoglobin solutions were heated to 60°C for 30 min, followed by centrifugation at 15,000 × g for 10 min. The hemoglobin concentration remaining in the supernatant was measured spectrophotometrically. Data are reported as mean ± standard deviation. Turkey-Kramer test was used for the statistical evaluation of the data.


Oxygen equilibrium curves of hemoglobins

The oxygen binding properties of the three different mouse hemoglobins, HbbS, Hbbmin and Hbbmaj are shown in Table 1 and Fig. 2. The mean P50, the partial oxygen pressure at which hemoglobin is 50%-saturated, for HbbS was significantly higher than that of Hbbmin and Hbbmaj (p < 0.001). The addition of 2 mM 2,3-DPG or 1 mM IHP shifted the OEC toward the right (Table 1). The Hill's n value was 2.45 to 2.59 for the mouse hemoglobins. The Hill's n value, which is an indication of hemoglobin subunit cooperativity, for mouse hemoglobins is slightly lower than the average value of 2.8-3.0 for normal human hemoglobin (Bunn and Forget, 1986). Riggs (1960) and Riggs and Herner (1962) reported that the alkaline Bohr effect was 0.9 in mice, which is much larger than that of human and larger mammals' hemoglobin. However, we determined the alkaline Bohr effect of purified HbbS, Hbbmin and Hbbmaj to be 0.69, 0.61 and 0.60, respectively (Table 1 and Fig. 3). Our results are consistent with those reported by Smith et al. (1966), who found a Bohr effect of 0.6. The Bohr factor of human hemoglobin is 0.6 (Bunn and Forget, 1986), therefore there is little deference in the Bohr effect between mouse and human hemoglobins.

Table 1

Oxygen affinity and Bohr factor of mouse hemoglobins


Fig. 2

The effect of pH on oxygen affinity of mouse hemoglobins. The slope of the curve between pH 6.5 and 7.5 is a measure of the alkaline Bohr effect. There were no significant differences between the slopes of the three mouse hemoglobins.


Oxygen equilibrium curves of red cell suspensions

The mean 2,3-DPG and P50 values in mouse RBC with HbbS/HbbS, HbbS/Hbbmin, Hbbmin/Hbbmin and Hbbmaj/Hbbmin mice are shown in Table 2. The mean 2,3-DPG concentration in HbbS/HbbS mice was significantly higher than that of mice with other hemoglobins. Although the differences in P50 values were not statistically significant, RBC from Hbbmaj/Hbbmin and Hbbmin/Hbbmin mice tended to have lower mean P50 values compared to HbbS/HbbS and HbbS/Hbbmin mice.

Table 2

Red blood cell P50 values, 2,3-DPG and hematocrit in mice with four different mouse hemoglobin phenotypes


Newton and Peters (1983) reported that RBC from mice with Hbbmaj/Hbbmin tend to have a lower P50 than RBC from mice with HbbS/HbbS. This is consistent with our results and is reasonable, because both the P50 for hemoglobin solution from Hbbmaj/Hbbmin RBC and the 2,3-DPG concentration in Hbbmaj/Hbbmin RBC were lower than those of HbbS/HbbS RBC in this study. In mice with HbbS/Hbbmin, the percentage of HbbS was significantly greater than Hbbmin and the P50 for the hemoglobin solution of HbbS was higher than that of Hbbmin. This could explain why RBCs from mice with HbbS/Hbbmin tended to have a higher P50 value than did mice with Hbbmin/Hbbmin.

Hbbmaj/Hbbmin mice have 80% Hbbmaj and 20% Hbbmin. The P50 for the Hbbmaj and Hbbmin were lower than that for HbbS, which explains why the P50 of RBC from Hbbmaj/Hbbmin mice was lower than that from HbbS/HbbS mice.

Newton and Peters (1983) also reported an increased hematocrit in Hbbmaj/Hbbmin mice. They postulated that this increase was the result of a lower RBC P50 in Hbbmaj/Hbbmin mice than in HbbS/HbbS mice, which caused a decrease in oxygen delivery to the tissues. In our study, we did not find a significant difference in the hematocrit values between Hbbmaj/Hbbmin and HbbS/HbbS mice, although the P50 of the Hbbmaj/Hbbminmice was lower than that of the HbbS/HbbS mice. In Hbbmin/Hbbmin mice, both the P50 and hematocrit were decreased, suggesting a decrease in oxygen transport.

Mechanical and heat stability of hemoglobins

Since this is the first report to examine the stability of various mouse hemoglobins by mechanical shake and heat denaturation methods, we used human HbA and HbS for comparison. As shown in Table 3, the k-values (Ohnishi et al., 1974), which represent the first order denaturation constants, indicated that mouse Hbbmaj was the least stable among all hemoglobins tested and similar to that of human HbS. On the other hand, mouse Hbbmin was most stable. The mechanical stability of HbbS was intermediate between human HbA and HbS, while that of Hbbmin was similar to HbA. In the heat stability tests, mouse Hbbmaj was the least stable among the three mouse hemoglobins tested. All mouse hemoglobins were less stable to heat denaturation than human HbA and HbS.

Table 3

Mechanical and heat stability of mouse and human hemoglobins



We thank Kazuhiko Adachi, Ph.D. for valuable advice in purifying hemoglobin.



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Kou Uchida, Michael P. Reilly, and Toshio Asakura "Molecular Stability and Function of Mouse Hemoglobins," Zoological Science 15(5), 703-706, (1 October 1998).
Received: 18 September 1997; Accepted: 1 May 1998; Published: 1 October 1998
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