Detection of an unstable murine hemoglobin

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

Pages 551-556

September 16, 1986

DETECTION OF AN UNSTABLE MURINE HEMOGLOBIN C. J. Wawrzyniak I and R. A. Popp

2

Iuniversity of Tennessee, Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831 2Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received August 4, 1986 ~ : 3H-leucine was used in vitro to label newly synthesized adult ~ and B globins in reticuloeytes removed from normal (Hba-b/Hba-b;Hbb-s2/Hbb-s2 and a-thalassemic (Hba-b2(th)/Hba-b;Hbb-s2/Hbb-s2) mice. The ratio of synthesis of B-s2major : B-sminor globins was 71 : 29 in retieulocytes from normal mice and 55 : 45 in reticuloeytes from a-thalassemie mice. The two B-globins are structurally identical except for a Val ÷ Glu substitution at position 60. Denaturation of these mouse hemoglobins in isopropanol indicated that the tetramer containing the B-s2major globin is unstable.

Unstable hemoglobins have been previously described for humans (1,2), but none have been reported for the laboratory mouse.

The unstable murine hemo-

globin mutation, Hbb-s2, used in this study was induced by ethylnitrosourea (3). The unstable property was recognized during studies on the synthesis of B-globin chains in reticulocytes of Hbb-s2/Hbb-s2 mice that carried an a-thalassemic allele at the ~-globin locus, Hba.

Hereditary murine a-thalassemias are

caused by deletion of the a-globin genes (4).

Due to the deficient synthesis

of ~-globin chains, B-globin chains are present in excess in the mouse as in ~-thalassemic humans (5).

Contrary to expectation we found that the amount of

and B chains was equal in retieulocytes from Hba-b2(th)/Hba-b;Hbb-s2/Hbb-s2 mice, and the ratio of B-s2major : B-sminor was also perturbed. In this report we describe experiments to determine the instability of B-s2major globin when it is subjected to denaturation by isopropanol.

MATERIALS AND METHODS Mice. Carriers of an ethylnitrosourea induced mutation at the Hbb-s locus (3) were brother-sister mated to produce the stock of homozygous Hbb-s2/Hbb-s2 mice used in this study. These mice were mated with the X-radiation-induced ~-thalassemia mutant, 352HB (6). Identification of mice that were heterozygous for ~-thalassemia and homozygous for the Hbb-s2 mutation (Hba-b/Hba-b2(th); Hbb-s2/Hbb-s2) was done by microscopic inspection of blood films for 0006-291X/86 $1.50 551

Vol. 139, Ne. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

anisocytosis and poikilocytosis (7) and the ~-globin alleles were classified by electrophoresis (described below). ElectroDhoresis and seannin~ densitometry. Separation and quantitation of hemoglobins was done as previously described (8). In vitro ~lobin synthesis. Globin chains were labeled with 3H-leucine and separated by carboxymethycellulose (CMC) column chromatography as previously described (8). Precipitation in isonroDanol. The following procedure is a modification from Carrell and Kay (12). Adult non-thalassemic mice were bled from the supraorbital sinus into 30 ml of 0.67 percent NaCI, 1.0 percent Na-citrate. The red cells were washed twice in 0.85 percent saline. The red cell pellet was lysed in 1.5 times its volume with water. The solution of lysed red cells was centrifuged at 20,000 xg for 30 min at 0°C to pellet the cell membranes and the supernatant will now be referred to as the hemolysate. The optical density at 575 nm of all hemolysates was adjusted to 0.58. Isopropanol solutions of 15, 16, 17, 18, 19, 20, 22 and 25 percent by volume were made in 0.1M Tris buffer, and the pH was adjusted to 7.4 with HCI. All isopropanol solutions were warmed to 37°C in a water bath prior to mixing with the hemolysate. In a graduated tube 74 ~I of hemolysate was mixed with 667 ~i buffered isopropanol. The reactions wore carried out at 37°C in a water bath for 20 min, then the mixture was centrifuged at 600 xg for 10 min at 5°C. The volume of the precipitate was measured using the graduation on the tube. The supernatant was analyzed for hemoglobin content by cellulose acetate electrophoresis, followed by quantitation of hemoglobin bands with a scanning densitometer. RESULTS The 3H-leucine-labeled globins present in reticulocytes of adult mice with different a-globin genotypes were separated by CMC column chromatography and quantitated.

The results shown in Figure IA represent data from non-

thalassemic mice (Hba-b/Hba-b;Hbb-s2/~-s2) and in IB data from ~-thalassemic mice (Hba-b2(th)/Hba-b;Hbb-s2/Hbb-s2).

Data from four separate experiments

were used to determine a ratio of 71 ~-s2major : 29 B-sminor ± 1.6, with an a:S ratio of 49 : 51 ± 1.2, in retieulocytes from non-thalassemie mice.

Data

from three separate experiments were used to determine a ratio of 55 S-s2major : 45 S-sminor ± 3.2, and an a:S ratio of 53 : 47 ± 3.2 in reticulocytes from -thalassemic mice.

Earlier studies had shown that the ratio of ~:S globin

synthesis was 43 : 57 in retiouloeytes from a-thalassemic mice homozygous for the Hbb-s allele (9). Hemolysates were obtained from erythrocytes of three groups of adult mice.

Each group of mice had the same ~-globin genotype but each group had a

different S-globin genotype.

The hemolysates were mixed with concentrations

of buffered isopropanol ranging from 15 to 25 percent.

552

At each isopropanol

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

t4" A 12-0.5

108-

,8- s2rnajor

-0,4 -0.5

f/ _sm,n ° o~,;

4-

S 2-

-~

r-or2¢h

-0.2

~.

li~j7 ~..~., ,<

-0.1

£ ¢

i

u =30" ~J

B

?

~26" !

22-

-0.7 -0.6 f4-

B-s2mejor -0,5

,o

-0.4 '

40

60 80 FRACTION NO.

-0,3

'~" t00

0.2

Figure I. Separation and quantitation of globins by carborymethylcellul-'~o~umn chromatography. The labeling of hemoglobin with 3H-leucine and the isolation of the a- and B-chains were performed as described in Materials and Methods. The results shown are: A, reticulocytes of nonthalassemic (Hba-b/Hba-b;Hbb-s2/Hbb-s2) mice, and B, reticulocytes of ~-thalassemic (Hba-b2(th)/Hba-b;/~h-s2/Hbb-s2) mice.

concentration tested, the volumes of precipitates formed decreased in the following order: Hbb-s2/Hbb-s2 > Hbb-s2/Hbb-s > Hbb-s/Hbb-s.

The amount of

precipitate that developed in hemolysates obtained from erythrocytes of Hbb-s2/Hbb-s2 and Hbb-s/Hbb-s mice mixed with buffered isorpopanol adjusted to 20 percent is shown in Table I.

There was 1.8 times more precipitate formed

from the Hbb-s2/Hbb-s2 hemolysate than from the Hbb-s/Hbb-s hemolysate. The quantity of each tetramer, ~2B2s2maj °r or a262 sminor, remaining in solution after incubation of the Hbb-s2/Hbb-s2 hemolysate in isopropanol was analyzed by eleetrophoretically separating these hemoglobins on a cellulose acetate plate and scanning the bands with a densitemeter.

In these

supernatants, where I O0 percent = S-s2major + B-sminor, the amounts of B-sminor present were 31, 33, 34 and 38 percent at isopropanol concentrations of 18,

553

Vol. 139, No, 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

19, 20 and 22 percent, respectively.

In addition,

the relative levels of

B-sminor in the supernatant increased if the reaction was allowed to incubate for 40 or 60 minutes longer. DISCUSSION AND CONCLUSION Reticulocytes of mice which are a-thalassemic and homozygous Hbb-s synthesize deficient quantities of ~-globin chains, where a:B is 43 : 57 (9). However, the ~:S ratio in reticulocytes from the ~-thalassemic mice used in this study was 53 : 47 as determined by separating the 3H-leucine-labeled chains by CMC chromatography.

This could be explained if one of the S-globin

chains coded for by the Hbb-s2 haplotype is unstable.

The difference presumably

resides in the S-globins because the relative quantities of S-s2major : S-sminor are different in reticulobytes of ~-thalassemic and non-thalassemic mice having the same S-globin genotype.

The less stable chain was identified as

the B-s2major by its relatively decreased amount in reticulocytes of a-thalassemic mice (Figure IB).

The lower stability of the S-s2major chain

subjects it to greater intracellular degradation by proteolysis.

Similar

proteolysis of the excess globin chain has been demonstrated in reticulocytes of thalassemic patients (13,14). The extent of the instability of the B-s2major chain was determined by measuring the volume of precipitation of globin that developed when hemolysates from mice with different S-globin genotypes were incubated in isopropanol. The organic solvent weakens the hydrogen bonding between the heme and the histidine residue at position 63 in the S-globin.

The least stable tetramers

precipitate because globin is less soluble than the heme-globin complex. Comparisons were made between hemolysates obtained from erythroeytes of adult Hbb-s/Hbb-s mice, which do not code for the ~-s2major chain, and Hbb-s2/Hbb-s2 mice, in which 70 percent of the S-globin is of the S-s2major type (8). Hemolysates from mice with S-sPmajor globin formed I .8 times more precipitate when incubated in buffered isopropanol adjusted to 20 percent than hemolysates that did not have S-s2major (Table I).

This demonstrated that

the heme bound to the fi-s2major chain is less stable.

554

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

TABLE 1:Denaturation of Mouse Hemoglobin in Isopropanol Hmmo~1obln GenotvDe ~g-b/Hba-b;~-s/Hbb-s Volume of precipitate (wl)a Relative amount of precipitatec

18.3 ± 1.8b

-Hba-b/Hba-b;Hbb-s2]~-s2

32.8 ± 0.4

1.0

1.8

aVolume of precipitate obtained from reacting 667 u i of 22 percent buffered isopropancl solution with 74 Wl of hemolysate for 20 min at 37°C. bstandard deviaOlon of the mean. CThe volume of precipitate in the control sample was normalized to 1.0.

Additional evidence that hemoglobin containing the S-s2maJor chain was precipitated in hemolysates o f ~ - s 2 / ~ - s 2

blood comes from analysis of the

supernatant after precipitation of the less stable hemoglobin.

The relative

quantity of S-sminor remaining in the supernatant increased as more precipitate was formed.

This observation indicates that the less stable

hemoglobin containing the ~s2major globin is denatured preferentially and removed from solution as the precipitate forms. In human hemoglobin Milwaukee there is a replacement of valine with glutamic acid at position 67, and it has been suggested that the carboxyl group of glutamic acid forms a salt bridge with iron and pushes the side chain of histidine out of the heme pocket making the molecule less stable (15,16).

A similar amino acid substitution located in an adjacent area of

the heme pocket (Val + Glu position 60) is involved in the unstable globin described here.

The demonstration of increased precipitation in isopropanol

suggests a similar perturbation of the conformational structure may occur in the heme pocket of this unstable murine S-globin. In ~-thalassemic mice, where ~-globin synthesis is deficient, the S-s2major chains that are not immediately assembled into the normal tetrameric state become denatured and are proteolyzed more rapidly than are normal S-sminor globin chains.

The hematological parameters of Hbb-s2/Hbb-s2 mice fall in

the normal ranges (8); furthermore, the spleen weight of these mice is normal 555

Vol, 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(80 ± 1.4 mg), which indicates that there is no hemolysis associated with this unstable hemoglobin.

The unstable property of the ~-s2major globin

chain becomes physiologically evident only when subjected to denaturating conditions,

such as isopropanol, or in a-thalassemic mice where the S-s2major

and the more stable S-sminor globin chains compete for a decreased amount of a-globin during the assembly of tetrameric hemoglobin molecules.

We thank R. Julian Preston, K. Bruce Jacobson and Diana M. Popp for carefully reviewing this manuscript. This research was supported jointly by NIH Predoctoral Training Grant in Genetics GM-7438 and the Office of Health and Environmental Research, U.S. Department of Energy under contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. The U.S. Government's right to retain a nonexclusive royalty-free license in and to any copyright covering the article, for governmental Durposes, is acknowledged. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Huehns, E. R. (1970) Annual Review of Medicine (DeGraff, A. C. and Creger, W. P., eds.), pp 157-178, Annual Reviews, Inc., Palo Alto. Bunn, H. F., Forget, B. G. and Ranney, H. M. (1977) "Hemoglobinopathies." Philadelphia: W. B. Saunders. Lewis, S. E., Johnson, F. M., Skow, L. C., Popp, D., Barnett, L. B. and Popp, R. A. (1985) Proc. Natl. Acad. Sci. USA 82, 5829-5831. Whitney, J. B., III, Martinell, J., Popp, R. A., Russell, L. B. and Anderson, W. F. (1981) Proc. Natl. Acad. Sci. USA 78, 7644-7647. Weatherall, D. J. and Clegg, J. B. (1981) "The Thalassemia Syndromes." Third Edition, Oxford: Blaekwell Scientific Publications. Russell, L. B., Russell, W. L., Popp, R. A., Vaughan, C. and Jacobson, K. B. (1976) Proc. Natl. Aead. Sci. USA 73, 2843-2846. Popp, R. A. and Enlow, M. K. (1977) Amer. J. Vet. Res. 38, 569-572. Wawrzyniak, C. J. and Popp, R. A. (1985) Dev. Biol. 112, 477-484. Martinell, J., Whitney, J. B., III, Popp, R. A., Russell, L. B. and Anderson, W. F. (1981) Proe. Natl. Acad. Sci. USA 78, 5056-5060. Rossi-Fanelli, A., Antonini, E. and Caputo, A. (1958) Biochim. Biophys. Aeta 30, 357-450. Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1966) J. Mol. Biol. 19, 91-108. Carrell, R. W. and Kay, R. (1972) Brit. J. Haematol. 23, 615-619. Shaeffer, J. R. (1983) J. Biol. Chem. 258, 13172-13177. ~Adams, J. G., III, Boxer, L. A. and Baehner, R. L. (1979) J. Olin. Invest. 63, 931-938. Gerald, P. S. and Efron, M. L. (1961) Proc. Natl. Acad. Sei. USA 47, 1758-1767. Carrell, R. W. and Winterbourn, C. C. (1981) Texas Reports on Biology and Medicine, Vol. 40 (Schneider, Charache and Schroeder, eds.), pp 431-446, The University of Texas, Austin.

556