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Spontaneous oxidation of the γ chain of the foetal haemoglobin component of cord blood
SHORT COMMUNICATIONS
495
by FLATMARK5. The product had a molar absorbance (dry weight basis; single determination; p H 7.0; /,--~ o.I) of 2.8.1o 4 M - l . c m -1 in the reduced form at 550 m/~, and gave a value of 1.2 for the ratio e55o ~lu (red)/e~so mu (OX). Samples were hydrolyzed with constant-boiling hydrochloric acid in evacuated ampules at IiO ° for periods of 24 and 48 h, and the hydrolysates analyzed with a Beckman-Spinco model I2oB automatic amino acid analyzer 6,7. The results are summarized in Table I. As m a y be seen, our data are at variance with those of PALI~US,but agree well with the composition proposed by 3{ARGOLIASHet al. It would seem improbable that the differences in the two sets of results are a reflection of sub-species differences. In the absence of direct evidence on this point, we have, however, taken the precaution of using hearts all taken from a single breed. This work was supported b y grants from the National Institutes of Health, U.S. Public Health Service (GM-o7317) and the National Science Foundation (GB 1556). We are indebted to the Campbell Soup Company for a generous gift of chicken hearts, and to Mr. D. H. BECKER of that Company for his help in effecting the necessary arrangements. We should like to thank Dr. W. KONIGSBERG for the use of his amino acid analyzer.
Department of Biochemistry, Yale University, New Haven, Conn. (U.S.A.)
SERGE N . VINOGRADOV HENRY A. HARBURY
I E..'X,IARGOLIASH, S. B. NEEDLEMAN AND J. \V. STEVVART,Acta Chem. Scand., 17 (1963) $25o. E..~IARGOLIASH, Brookhaven Syrup. Biol., (BNL738 (C-34)), 15 (1962) 266. 3 S. PAL~US, Acta Chem. Scand., 18 (1964) 1324. 4 S. ]~ALt~US, Acta Chem. Scand., 14 (196o) 1743. 5 T. FLATMARK, Acta Chem. Scand., 18 (1964) 1517. 6 S. ~fOORE, D. H. SPACKMAN AND W. H. STEIN, Anal. Chem., 3 ° (1958) 1185. 7 D. ]-I. SPACKMAN, W. H. STEIN AND S. ~V[OORI~,Anal. Chem., 3 ° (1958) 119o.
Received August 23rd, 1965 Biochim. Biophys. Acta, 115 (1966) 494-495
BBA 23 I91
Spontaneous oxidation of the ~ chain of the foetal haemoglobin component of cord blood In an attempt to study the incidence of fast-moving haemoglobins in the blood of Dutch neonates, we have recently examined one hundred samples of cord-blood by means of starch-gel electrophoresis in Tris buffer at p H 8.3. Though we did not find any fast-moving haemoglobins, we came across the interesting phenomenon of partial spontaneous oxidation of the 7 chain of the foetal haemoglobin component, leading to the formation of 1VIetHb. This we consider to be worth recording. When electrophoresis was performed on the blood samples in the form of an HbO2 solution, the majority of specimens showed, apart from the normal Hb-A and A b b r e v i a t i o n s : H b - A , a d u l t h a e m o g l o b i n ; H b - F , foetal h a e m o g l o b i n ; H b O 2, o x y h a e m o globin; M e t H b , m e t h a e l n o g l o b i n .
Biochim. Biophys. Acta, 115 (1966) 4 9 5 - 4 9 7
496
SHORT COMMUNICATIONS
H b - F components, a third fraction which had a mobility slightly slower than Hb-S (see Fig. I). Those samples, which did not show this third fraction on first examination, invariably showed it on subsequent examination, after storage at 3 '~ over a period of at least 3 days. However, this third fraction could not be detected after converting the haemolysates into the cyanmethaemoglobin form. Mixing equal amounts of the HbO2 and MetHb forms of cord blood, the abnormal fraction could be demonstrated more clearly (Fig. 2). The MetHb should be dialysed thoroughly against water. By electrophoresis, it was proved that this third fraction was not identical with the normal MetHb of either Hb-A or H b - F (see Fig. 2). In order to investigate this ab-
Hb-A Hb-F Hb-S and ,,Third f r a c t i o n "
Start 1
2
3
Heterozygous Hb-$
4
5
6
Fig. I. Starch-gel electrophoresis at pH 8.3 of 6 cord-blood samples in comparison with heterozygous Hb-S.
~IbO2"A ~lbO2-F MetHb-A Third f r a c t i o n " MetHb-F
Start Adult blood oxidised
Mixture Cord blood cord blood ox idised oxy and me~ . . . . form
Fig. 2. Starch-gel electrophoresis at pH 8.3. Biochim. Biophys. Acla, 115 (1966) 495 497
Cord blood oxy form
497
SHORT COMMUNICATIONS
normal fraction, a mixture of Hb02 and MetHb of cord-blood was chromatographed on CM-cellulose. Four fractions were obtained, the same as appeared on electrophoresis (Fig. 2). Spectra of these fractions were measured in the Beckman DB spectrophotometer. The spectrum of the first fraction was that of pure HbO 2 and that of the last fraction was identical with the spectrum of pure MetHb. The electrophoretic mobility and the ultraviolet spectra made it likely that the first fraction was HbO2-A and the last fraction MetHb-F. The second fraction showed a spectrum of HbOs slightly contaminated with MetHb. This fraction was thought to be a mixture of HbO2-F and MetHb-A, and was proved to be so by measuring the spectrum of the first and last tubes. The first tubes showed a spectrum of HbO~ whereas the last tubes had a spectrum very like pure MetHb. The spectrum of the third and unknown fraction is shown in Fig. 3. It is a mixed spectrum of HbO 2 and MetHb and is very similar to the spectrum obtained from haemolysates of patients with an abnormal MetHb, such as the Hb-Molaenburg carriers 1. Oxidation of the abnormal fraction with K3Fe(CN)G gave the normal MetHb-F. We therefore assume that the third fraction is a partial oxidation product of the Hb-F molecule. It is not known which of the chains is oxidised. Because similar experiments with Hb-A do not give rise to an abnormal fraction, it seems very likely that the oxidation occurs at the haem group of one or both 7 chains of the Hb-F molecule.
g 700
650
600
I
I
I
550
500
450
Wavelength (mlJ)
Fig. 3. S p e c t r u m of the " t h i r d fraction".
One of us, F.S.B., is grateful to the Netherlands Bureau for International Technical Assistance (Nebuta) for the award of a fellowship. This investigation was supported in part by a research grant, No. HE-o653I-O3, from the U.S. Public Health Service.
Department of Pediatrics, The State University, Groningen (The Netherlands)
F. S. BoI-DoKu* C. PIK
I O. TONZ, H. A. SIMON AND W. HASSELFELD,Schweiz. Meal. Wochschr., 92 (1962) 131t.
Received August 25th, 1965 * Present address: Central Clinical L a b o r a t o r y Korle Bu Hospital, Accra, Ghana.
Biochim. Biophys. Acta, I15 (I966) 495-497
495
by FLATMARK5. The product had a molar absorbance (dry weight basis; single determination; p H 7.0; /,--~ o.I) of 2.8.1o 4 M - l . c m -1 in the reduced form at 550 m/~, and gave a value of 1.2 for the ratio e55o ~lu (red)/e~so mu (OX). Samples were hydrolyzed with constant-boiling hydrochloric acid in evacuated ampules at IiO ° for periods of 24 and 48 h, and the hydrolysates analyzed with a Beckman-Spinco model I2oB automatic amino acid analyzer 6,7. The results are summarized in Table I. As m a y be seen, our data are at variance with those of PALI~US,but agree well with the composition proposed by 3{ARGOLIASHet al. It would seem improbable that the differences in the two sets of results are a reflection of sub-species differences. In the absence of direct evidence on this point, we have, however, taken the precaution of using hearts all taken from a single breed. This work was supported b y grants from the National Institutes of Health, U.S. Public Health Service (GM-o7317) and the National Science Foundation (GB 1556). We are indebted to the Campbell Soup Company for a generous gift of chicken hearts, and to Mr. D. H. BECKER of that Company for his help in effecting the necessary arrangements. We should like to thank Dr. W. KONIGSBERG for the use of his amino acid analyzer.
Department of Biochemistry, Yale University, New Haven, Conn. (U.S.A.)
SERGE N . VINOGRADOV HENRY A. HARBURY
I E..'X,IARGOLIASH, S. B. NEEDLEMAN AND J. \V. STEVVART,Acta Chem. Scand., 17 (1963) $25o. E..~IARGOLIASH, Brookhaven Syrup. Biol., (BNL738 (C-34)), 15 (1962) 266. 3 S. PAL~US, Acta Chem. Scand., 18 (1964) 1324. 4 S. ]~ALt~US, Acta Chem. Scand., 14 (196o) 1743. 5 T. FLATMARK, Acta Chem. Scand., 18 (1964) 1517. 6 S. ~fOORE, D. H. SPACKMAN AND W. H. STEIN, Anal. Chem., 3 ° (1958) 1185. 7 D. ]-I. SPACKMAN, W. H. STEIN AND S. ~V[OORI~,Anal. Chem., 3 ° (1958) 119o.
Received August 23rd, 1965 Biochim. Biophys. Acta, 115 (1966) 494-495
BBA 23 I91
Spontaneous oxidation of the ~ chain of the foetal haemoglobin component of cord blood In an attempt to study the incidence of fast-moving haemoglobins in the blood of Dutch neonates, we have recently examined one hundred samples of cord-blood by means of starch-gel electrophoresis in Tris buffer at p H 8.3. Though we did not find any fast-moving haemoglobins, we came across the interesting phenomenon of partial spontaneous oxidation of the 7 chain of the foetal haemoglobin component, leading to the formation of 1VIetHb. This we consider to be worth recording. When electrophoresis was performed on the blood samples in the form of an HbO2 solution, the majority of specimens showed, apart from the normal Hb-A and A b b r e v i a t i o n s : H b - A , a d u l t h a e m o g l o b i n ; H b - F , foetal h a e m o g l o b i n ; H b O 2, o x y h a e m o globin; M e t H b , m e t h a e l n o g l o b i n .
Biochim. Biophys. Acta, 115 (1966) 4 9 5 - 4 9 7
496
SHORT COMMUNICATIONS
H b - F components, a third fraction which had a mobility slightly slower than Hb-S (see Fig. I). Those samples, which did not show this third fraction on first examination, invariably showed it on subsequent examination, after storage at 3 '~ over a period of at least 3 days. However, this third fraction could not be detected after converting the haemolysates into the cyanmethaemoglobin form. Mixing equal amounts of the HbO2 and MetHb forms of cord blood, the abnormal fraction could be demonstrated more clearly (Fig. 2). The MetHb should be dialysed thoroughly against water. By electrophoresis, it was proved that this third fraction was not identical with the normal MetHb of either Hb-A or H b - F (see Fig. 2). In order to investigate this ab-
Hb-A Hb-F Hb-S and ,,Third f r a c t i o n "
Start 1
2
3
Heterozygous Hb-$
4
5
6
Fig. I. Starch-gel electrophoresis at pH 8.3 of 6 cord-blood samples in comparison with heterozygous Hb-S.
~IbO2"A ~lbO2-F MetHb-A Third f r a c t i o n " MetHb-F
Start Adult blood oxidised
Mixture Cord blood cord blood ox idised oxy and me~ . . . . form
Fig. 2. Starch-gel electrophoresis at pH 8.3. Biochim. Biophys. Acla, 115 (1966) 495 497
Cord blood oxy form
497
SHORT COMMUNICATIONS
normal fraction, a mixture of Hb02 and MetHb of cord-blood was chromatographed on CM-cellulose. Four fractions were obtained, the same as appeared on electrophoresis (Fig. 2). Spectra of these fractions were measured in the Beckman DB spectrophotometer. The spectrum of the first fraction was that of pure HbO 2 and that of the last fraction was identical with the spectrum of pure MetHb. The electrophoretic mobility and the ultraviolet spectra made it likely that the first fraction was HbO2-A and the last fraction MetHb-F. The second fraction showed a spectrum of HbOs slightly contaminated with MetHb. This fraction was thought to be a mixture of HbO2-F and MetHb-A, and was proved to be so by measuring the spectrum of the first and last tubes. The first tubes showed a spectrum of HbO~ whereas the last tubes had a spectrum very like pure MetHb. The spectrum of the third and unknown fraction is shown in Fig. 3. It is a mixed spectrum of HbO 2 and MetHb and is very similar to the spectrum obtained from haemolysates of patients with an abnormal MetHb, such as the Hb-Molaenburg carriers 1. Oxidation of the abnormal fraction with K3Fe(CN)G gave the normal MetHb-F. We therefore assume that the third fraction is a partial oxidation product of the Hb-F molecule. It is not known which of the chains is oxidised. Because similar experiments with Hb-A do not give rise to an abnormal fraction, it seems very likely that the oxidation occurs at the haem group of one or both 7 chains of the Hb-F molecule.
g 700
650
600
I
I
I
550
500
450
Wavelength (mlJ)
Fig. 3. S p e c t r u m of the " t h i r d fraction".
One of us, F.S.B., is grateful to the Netherlands Bureau for International Technical Assistance (Nebuta) for the award of a fellowship. This investigation was supported in part by a research grant, No. HE-o653I-O3, from the U.S. Public Health Service.
Department of Pediatrics, The State University, Groningen (The Netherlands)
F. S. BoI-DoKu* C. PIK
I O. TONZ, H. A. SIMON AND W. HASSELFELD,Schweiz. Meal. Wochschr., 92 (1962) 131t.
Received August 25th, 1965 * Present address: Central Clinical L a b o r a t o r y Korle Bu Hospital, Accra, Ghana.
Biochim. Biophys. Acta, I15 (I966) 495-497