Mouse hemoglobin during gestation Absence of fetal hemoglobin

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M O U S E H E M O G L O B I N DURING GESTATION A B S E N C E OF FETAL H E M O G L O B I N ROBERT MARTIN ZULKER ,,&m~ ( "ancer Re~'earch In.~tltute.

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(Received October 9th, 1981)

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A "fetal hemoglobin' has been reported to exist during mouse gestation. Investigations using CMC chromatography, starch gel electrophoresis or is(uelectric focusing have shown a hemoglobin band from fetal tissues, and blood was obtained which was different from the adult hemoglobin and designated as 'fetal hemoglobin'. In the current stud)' isoelectric focusing was used to study the hemoglobins existing in the tissues and blood during fetal and neonatal development and the results suggest there is no 'fetal hemoglobin' present during gestation. It appears that the hemoglobin designated as 'fetal' in our laborato D was a methemoglobin formed by an incomplete reaction of KCN with the hemoglobin. The additional hemoglobin bands which were obtained from fetal liver or neonatal spleen tissues appeared to be a modified adult hemoglobin.

Introduction Embryonic nucleated erythrocytes derived from the yolk-sac blood islands produce three forms of embryonic hemoglobins. The globin chains of these hemoglobins are designated x , y 2 for E l, ¢~,y, for Ell and c%z e for E m [1-3]. After day 12 of fetal development, the fetal liver releases non-nucleated erythrocytes into the circulation producing primarily adult hemoglobins. The relative increase in non-nucleated erythrocytes and decrease in yolk sac-derived nucleated erythrocytes results in a daily change in the percentage of different hemoglobin types. Later during gestation two other hemopoietic organs, spleen and bone marrow, become active in releasing erythrocytes into the circulating blood [4,5]. A "fetal hemoglobin' has been reported to occur in the mouse during gestation [6-8]. This hemoglobin is defined as being produced primarily during fetal development from hemopoietic tissue ()I67-4H38/x2 ,oo(x} 0(x10/$o2.75 , 19x2 Flse'.ier Biomedical Prc~,~,

such as liver, spleen or bone marrow. Kraus et al. [6] reported the neonatal spleen and fetal liver of the C 5 7 B L / c u m mouse had a 'fetal hemoglobin" differing in electrophoretic mobility from adult and embryonic hemoglobins. Schalekamp et al. [7] also observed 'fetal hemogh>bins" in the fetal liver of the Porton white Swiss mouse. Wu et al. [[4] used analytical and preparative isoelectric focusing to detect and purify a 'fetal hemoglobin" [8]. Even with these published reports there remains a controversy on the existence of the 'fetal hemoglobin' in the mouse [9]. Fantoni et al., Barker and Whitney showed there was no specific 'fetal hemoglobin' [2,5,10]. Whitney el al. postulated that the 'fetal hemoglobin' band that Kraus found in the neonatal spleen was a modified adult hemoglobin, and Morton suggested the bands may have occurred by S-S linkages [11,12]. This paper uses the sensitive IEF technique to study the hemoglobins found during fetal development and resolve the existence of the 'fetal hemo-

213 globins' found in other investigations. The results of this study suggest there is no 'fetal hemoglobin'. The hemoglobin described by Schalekamp et al. and Kraus et al. appears to be a modified hemoglobin which is electrophoretically similar to Al~. of humans [6,7]. The 'fetal hemoglobin' described by Wu et al. appears to be methemoglobin formed by incomplete reaction of hemoglobin in the KCN cyanonaethemoglobin method [8]. Materials and Methods

Animals. Adult C57BL/6 mice were obtained from the Mammalian Genetics Branch. National Cancer Institute. The fetal age was calculated by observation of vaginal plugs and considered to be zero-day old on the date of plug discovery. Additional classification of the fetal age was made by fetal size and electronic volume analysis [13]. Preparation of hemoglobins. Pregnant mice were killed by cervical dislocation. The uteri were removed and rinsed in cold Dulbecco's phosphatebuffered saline. The fetuses were isolated, rinsed and placed in cold phosphate-buffered saline containing 10 U / m l heparin. The fetal blood samples were collected by incision of the jugular vein of fetuses. Cell volume distributions of fetal blood were then measured by a Coulter Channelyzer, Model H4 (Coulter Electronics, Hialeah, FL)[14]. Hemoglobin was obtained by washing erythrocytes three times with phosphate-buffered saline and the cell membranes were broken by adding three volumes of lysing medium [14]. Cell debris was removed by centrifugation at 20000 × g for 20 min. The standard procedure in preparing hemoglobin for IEF was to mix 20/tl hemoglobin with 5~1 1M KCN and 5p.l 10% ampholine (pH 6 8). Between 2-5 p.l of this solution was added to the 3 mm polyacrylamide disc gels to achieve a concentration suitable for scanning at a 416 nm wavelength. Modifications of this procedure occurred in experiments which involved old KCN solutions and K3Fe(CN)6 oxidation. The old KCN was contained in either lysing solution ( 10 mM) [ 14] or stored as a 0.1 M stock solution at 4°C for 1-4 weeks. To partially oxidize the hemoglobins. 5 pA K3Fe(CN)6 (0.5.-0.05 mM) solution was added to the hemoglobin in place of KCN [15].

Spleen and liver were obtained from fetal, neonatal and adult animals. Hemoglobin was obtained by two methods. Initially the tissue was gently pushed through a 60- 80 stainless steel mesh screen and then washed in cold phosphate-buffered saline. The cells were lysed and the hemoglobin was obtained as previously described. Secondly. the tissues were homogenized with lysing medium and then hemoglobin was obtained as described. lsoelectric focusing. Analytical isoelectric focusing was performed in 3 mm internal diameter tubes using 4% polyacrylamide gels and pH 6 - 8 ampholines (LKB) [16]. The gels were prefocused at 0.15 W / g e l for 0.5h after which the samples were layered on the gel. lsoelectric focusing conditions consisted of a constant 0.15 W / g e l at a temperature of 4°C for 3 h. Results and Discussion

Three embryonic hemoglobins E l, Eli and Eln derived from yolk-sac cells and one adult hemoglobin derived from liver cells are observed at the 14th day of gestation (Fig. la). The addition of potassium ferricyanide K sFe(CN) 6 to adult mouse hemoglobin results in the partial oxidation of the hemoglobin, producing two intermediate bands and one methemoglobin band between the adult hemoglobin and Eli bands (Fig. ib). In a similar study on the partial oxidation of human hemoglobin by K3Fe(CN) 6, these bands were designated to be intermediate bands. IB I and IBEI (a 2' f13- , a.~+fl2- ) and m e t h e m o g l o b i n ( a s ' f13~ )[151. In preparing the hemoglobins for IEF, either lysing solution containing 10 mM KCN was used or 10 mM KCN was added prior to IEF from a stock solution of 1- 4-week-old 0.1 M KCN. These KCN solutions resulted in the incomplete cyanomethemoglobin formation and partial oxidation of the hemoglobins. The resulting hemoglobin pattern shows a preferential increase in methemoglobin and only one intermediate band instead of the two found with KsFe(CN) 6 (Fig. Ic). The methemoglobin and E H hemoglobins appear as either one broad band or as a doublet band. This close proximity of the methemoglobin to the E H band suggested that the band was hidden in other investigations which used less sensitive biochemical separation procedures. The

214

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pH6

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Et Fig. I. llemoglobin changes after potassium ferricvanide oxidation and incomplete KCN reduction. Hemoglobin from 14-davold fetuses (a) was reacted with 0.05 mM K ~F~.((N)~, (b), or 10 mM old K('N (c). K.~F'~.(('N),,, oxidation of the adult hemoglobin resulted in the appearance of three nev.. bands between E N and A. The,, were designated methemoglobin, intermediate I and intermediate II. The incomplete reaction of adult hemoglobin b,, old KCN resulted in one intermediate and one methemoglobin band. The spectrophotometric scans 'a,ere made at 416 mn of the isoeleclric-focused hemoglobin in pol~,acrvlamide gels between pH 6 and 8.

simultaneous appearance of two bands correlating to the release of liver-derived cells suggested one band was 'fetal' and one was adult. This would be consistent with other animal systems in which both adult and 'fetal hemoglobins' are derived from the fetal liver. In a previous publication by Wu et al. [8], antibody production and purification against the hemoglobins in the region occupied by

E n and methemoglobin was successful because subsequent enrichment of the antibody removed the contaminating component of the adult antibody leaving only specificity for embryonic E n hemoglobin. This antibody showed specificity against the band designated as 'fetal' but in actuality the activity was against the embryonic E H hemoglobin. These data suggest the 'fetal' mouse hemoglobin previously reported by Wu et al. [8] was due to oxidized hemoglobin and incomplete cyanomethemoglobin reduction by KCN. Multiple hemoglobins occurring during mouse gestation have been described by numerous investigators. "Fetal hemoglobins' have been designated by Krause et al. (starch gel electrophoresis) [6], Schalekamp et al. (CMC chromatography) [7] and Wu et al. (IEF) [8]. The 'fetal hemoglobin' described by Schalekamp et al. [7] was eluted in front of HbA on cation-exchange chromatography and the one described by Kraus et al. was separated as a 'fast' band on starch gel electrophoresis. Using IEF, hemoglobins derived from the spleen or liver show an additional band with a pl slightly lower than the adult band. This band, with a decreased pl relative to the adult hemoglobin band, correlates with bands reported by Kraus et al. [6] and by Schalenkamp et al. [7]. These 'fetal hemoglobins' appeared only in the fetal and neonatal tissues and not in the circulating blood, suggesting either a differential switch mechanism of hemoglobin production occurred in the organs or a modification of the hemoglobin occurred during the tissue preparation procedure. Investigations in our laboratory into the tissue-derived hemoglobins revealed that adult hemoglobin was almost completely converted into this new band at 4°C within 2 weeks (Fig. 2). A similar conversion is obtained if the sample is incubated overnight at 25°C. The mixing of neonatal liver or spleen homogenate with adult blood also resulted in a conversion of adult hemoglobin to a new hemoglobin with a lower pl value. This increase in quantity with time or temperature of the band having a lower pl than the adult hemoglobin suggests the environment is responsible for the conversion. The data from this study suggest these "fetal hemoglobins' are artifacts which occurred in the handling of the hemoglobins prior to the separation procedure.

215

Acknowledgements

HEMOGLOBIN CONVERSION a

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pH6 @25"C

This work was s u p p o r t e d by P H S grant n u m b e r I R 0 1 C A 24165 a w a r d e d b y t h e N a t i o n a l C a n c e r Institute.

We

thank

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Whittington

and

A n d r e w Tershakovec for their valuable assistance in this s t u d y , a n d t h e h e l p f u l d i s c u s s i o n o f Drs. R. L e i f a n d B. C a m e r o n .

References ~.

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Fig. 2. Changes in liver-derived hemoglobin p/ values during storage. Hemoglobin was obtained by homogenizing the liver and allowing it to sit at either 25°C overnight or at 4°C for 2 weeks. Spectrophotometric scans were made at 416 nm of the isoelectric-focused hemoglobin in polyaco'lamide gels between pH 6 and 8. (a) I day 25°C. (b) I week 4°C. (c) 2 weeks 4':('.

d l i n g o f t h e h e m o g l o b i n s p r i o r t o the s e p a r a t i o n procedure.

I Fantoni, A., De La Chapelle, A. and Marks, P.A ( 19691 J. Biol. Chem. 244, 675-681 2 Fantoni. A., Bank. A. and Marks, P.A. (1967) Science 157, 1327--1329 3 Popp, R., Bradshav,, B.S. and llirsch, (i.P. (1979) in ('cllular and Molecular Regulation of Hemoglobin Switching (Stamatoyannopoulos, C. and Nienhuis, A., eds.), pp. 227235. Grune and Stratton, New York 4 Russell, E.S. and McFarland, EC. (1974) in Annals of the New York Academy of Sciences (Kitchen, A. and Boyer, S., eds.), Vol. 241, pp. 25-38, New York 5 Barker, J.E. (1968) Dev. Biol, 18, 14-19 6 Kraus, L.M., Rasad, A., Ohba. Y. and Woodhead, AP. (1974) in Annals of the New York Academy of Sciences (Kitchen, A. and Boyer, S., eds.). Vol. 241. pp. 683-690 7 Schalekamp, M., Harrison. P.R and Paul, J. (1975) J. Embr3,'ol. Exp. Morphol. 34. 355-371 8 Wu, N., Sikkema. I)A. and Zucker, RM. (1978) Bk~:him Biophys. Acta 536, 306-311 9 Kitchen, H. and Brett, I. (1974) in Annals of New York Academy of Sciences (Kitchen, A. and Boyer, S., eds.), Vol. 241, pp. 653-671 I0 Whitney, JB. (1977) ('ell 12, 863-871 I I Whimey, J.B.. III., McFarland, EC. and Russell, E.S. (1978) Dcv. Biol. 65, 233-239 12 Morton. J.R (1966) (Jen. Rcs. Carnb. 7, 76-85 13 Zucker, R.M, (1970) J. ('ell Physiol. 75, 241- 251 14 Rovera, (i., Abramczuk. J. and Surrey, S. (1977) FEBS Lett. 81. 366-37O 15 Tomoda, A. and Yoncyama, Y. (1979) Biochim. Biophys. Acta 581, 128--135 16 Righetti, P.G. and Dr~sdalc, J.W. (19761 Isoclectric Fc~'using, pp, 440- 465, American Elsevier, Nev. York