- Email: [email protected]
Some observations about the heterogeneity of haemoglobin in aluminum oxide chromatography
57 °
SHORT COMMUNICATIONS
VOL. 2 0 (1956)
Some observations about the heterogeneity of haemoglobin in aluminum oxide chromatography The heterogeneity of normal adult haemoglobin and of some a b n o r m a l haemoglobins is reported by several workers using different methods, for instance alkali d e n a t u r a t i o n 1, salting out s, chrom a t o g r a p h y on Amberlite IRC 5 o3,4, p a p e r electrophoresis 5 and c h r o m a t o g r a p h y on AIzO36,L Using the AI,O 3 column, VAN FOSSAN8 succeeded in fractionating H b of both adult and fetus into three distinct components. As this method seemed suitable for detecting the heterogeneity of H b some experiments were carried out to check the occurrence in blood of the different comp o n e n t s postulated. The results of this research are reported here. The haemoglobins used (A, F, 13 and C) have been described elsewhere 8. The preparation was carried out as reported earlierg; b u t the haemoglobin solutions (containing xo g per cent of COHb) were never dialysed, except in a few special cases described below. Two samples of a l u m i n i u m oxide suitable for c h r o m a t o g r a p h i c analysis were used, one obtained from Merck and one from the British Drug Houses Inc. (13.D.H.). The general procedure was similar to those described by VAN FOSSAN~: The A1203 was poured into the tube (diameter o.9 to 2. 5 cm × 5 to 2o cm) as a thick aqueous suspension and allowed to settle. Small a m o u n t s of C O H b (5-5 ° mg) were p u t on the column and development was carried out with distilled water. The p H of the eluate fractions collected varied between 9.5 and i o . I . Two red water-mobile bands descended in the column close to each other, while a third one which was immobile at the top was eluted with o.35 % ammonia. The a m o u n t of H b in each fraction was determined with a Beckman s p e c t r o p h o t o m e t e r at 5700 A. The results of the fractionation of various h u m a n haemoglobins (A, I3, C and H b from cord blood) are given in Table I. Since a relatively large deviation exists for the a m o u n t s of the fractions I and II, it will be clear t h a t no significant differences exist between the four haemoglobins studied. I t will be noted t h a t the ratio between the three fractions is quite different from t h a t reported by VAN FOSSAN, w h o found average values of 4 8 % (I), 3°/0 (II) and 49% ( I I I ) for adult Hb. These differences m a y be explained by differences in the batches of AlzO v though when B.D.H. and Merck aluminium oxide was used a p p r o x i m a t e l y similar results were obtained (Table I). The influence of dialysis on the H b solutions is remarkable. W h e n the COHb solution was dialysed against distilled water at 4 ° C for 96 hours the a m o u n t of the ammonia-mobile fraction decreased from 19% to 8 % , while moreover only one water-mobile fraction was observed (Table I). The effect of dialysing the H b solutions against 0.35 % a m m o n i a (45 hours at 4 ° C and 3 h o u r s against distilled water in order-to remove the ammonia) is still greater: only 5 % of the total a m o u n t of h a e m o g l o b i n remained at the top of the column (Table I). In some experim e n t s the dialysate was concentrated by freeze-drying and recombined in a threefold equivalent a m o u n t with the dialysed H b solution. The chromatographic separation showed an increase in the a m o u n t of the a m m o n i a - m o b i l e fraction (from 5% before to 2I % after the recombination), TABLE I CHROMATOGRAPHY
A llO, used
Merck BDH BDH 13DH BDH BDH BDH BDH 13DH BDH
OF
HAEMOGLOBIN
ON
Pr etreatrnent
---
---96 h dialysis against distilled water 8 h dialysis against o.35 % N H 3 45 h dialysis against 0.35 % N H 3 45 h dialysis against 0.35 % N H v recombined with a threefold equivalent a m o u n t of the dialysate 45 h dialysis against 0.35 % NH3, recombined with a threefold equimolar a m o u n t of G S H
AIaO a UNDER
Hb used
A A B C F A A A A A
VARYING
CONDITIONS
.Pem~,¢agt o! tke diOtrent ]raaions I
II
III
25 35 ± 5 (n = 8) 33 27 3~
52 46 :t: 5 (n = 8) 36 5z 42 92 85 95 79
23 19 ± 2 (~ = s) 31 2I 27 8 15 5 2i
8i
I9
voL. 20 (1956)
SHOAT COMMUNICATIONS
571
while in s o m e e x p e r i m e n t s two water-mobile fractions were observed. As g l u t a t h i o n e (GSH) is t h e m a i n c o m p o n e n t in t h e dialysate, similar e x p e r i m e n t s were carried o u t b y r e c o m b i n i n g t h e dialysed H b solution w i t h a threefold e q u i m o l a r a m o u n t of GSH. T h e r e s u l t s are q u i t e similar to those o b t a i n e d with t h e c o n c e n t r a t e d di70 a l y s a t e (Table I). F r o m t h e s e d a t a it m a y be c o n c l u d e d t h a t G S H is a p r o m i n e n t factor in t h e resolution of h a e m o g l o b i n into t h r e e c o m p o n e n t s . H o w e v e r similar results were o b t a i n e d on e x a m i n a t i o n of h a e m o g l o b i n solutions with ._gso different p H ' s (obtained b y slowly a d d i n g v e r y small a m o u n t s of hydrochloric acid). T h e d a t a of Fig. i show t h a t a decrease of t h e p H from 8.8 to 6.0 g a v e a two- to threefold increase in o t h e a m o u n t of t h e a m m o n i a - m o b i l e fraction. E This increase is still g r e a t e r w h e n c o n c e n t r a t e d "r1,0o dialysate or G S H was also a d d e d (Fig. i). z P r e t r e a t m e n t of t h e a l u m i n u m oxide with distilled w a t e r e x e r t e d a g r e a t influence on t h e p e r c e n t a g e of t h e a m m o n i a - m o b i l e fraction. I n s o m e e x p e r i m e n t s s m a l l c o l u m n s (0.9 × 5.0 cm) filled with AltO s were w a s h e d with large a m o u n t s of distilled w a t e r a n d t h e n used for c h r o m a t o g r a p h i c analyses. It w a s found t h a t t h e total q u a n t i t y of h a e m o g l o b i n , I 1 r e m a i n e d a t t h e t o p of t h e c o l u m n after a 6 7 8 pH p r e t r e a t m e n t With 15oo to 3ooo ml of distilled Fig. 1. T h e influence of t h e p H . o f t h e H b solution water. I n t h o s e cases t h e p H of t h e e l u a n t on t h e percentage of t h e a m m o n i a - m o b i l e fraction. was decreased f r o m 9.5 to 7.2 to 6.8. • • without addition; O O recombined F r o m t h e s e e x p e r i m e n t s it will be clear w i t h t h e c o n c e n t r a t e d dialysate; × x recomt h a t t h e s e p a r a t i o n of h a e m o g l o b i n into t h r e e bined w i t h GSH. I n all cases a d j u s t m e n t of t h e c o m p o n e n t s b y a l u m i n i u m oxide c h r o m a t o p H w i t h HCL g r a p h y is d e p e n d e n t on different e x p e r i m e n t a l conditions. I t s e e m s v e r y e a s y to c o n v e r t one c o m p o n e n t into t h e o t h e r b y slight e x p e r i m e n t a l alterations. T h e effect of small a m o u n t s of hydrochloric acid a n d of g l u t a t h i o n e in increasing t h e p e r c e n t a g e of t h e a m m o n i a - m o b i l e fraction m a y be explained b y a s s u m i n g t h a t s m a l l a m o u n t s of AltO * are c o n v e r t e d into AI(OH)s. This a l u m i n i u m h y d r o x i d e is able to absorb large a m o u n t s of haemoglobin TM. T h e s a m e e x p l a n a t i o n m a y be valid for t h e striking effect of w a s h i n g t h e A l t O s c o l u m n w i t h distilled w a t e r ; it m a y be t h a t larger a m o u n t s of AlsO a are c o n v e r t e d into AI(OH)a, t h u s a c c o u n t i n g for t h e high c a p a c i t y of s u c h a c o l u m n for a b s o r b i n g h a e m o g l o b i n . W e believe, therefore, t h a t t h e a p p a r e n t h e t e r o g e n e i t y of h a e m o g l o b i n as s h o w n b y a l u m i n i u m oxide c h r o m a t o g r a p h y is d u e to e x p e r i m e n t a l c o n d i t i o n s ; t h e use of this m e t h o d for p r o v i n g h a e m o g l o b i n h e t e r o g e n e i t y is strictly limited. H. K. PRINS Department o/Pediatrics, University o/Groningen (The Netherlands) T . H . J . HUZSMAN 1 R . BRXNKMA~, A. WILDSCHUT AND A. WITTERMAN, J. Physiol., 80 (1934) 377. t j . ROCHE AND Y. DERRIEN, Le Sang, 24 (1953) 97. • M. MORRISON AND J. CooK, Science, 12o (1955) 920. t H. K. PRINS AND T. H. J. HUISMAN, Nature (in t h e press). s H. G. KUNKEL AND G. WALLENIUS, Scie~,ce, 122 (I955) 288. • D. D. VAN FOSSAN, Federation Prec., 13 (1954) 156; R e p o r t No. 7; P r o j e c t No. 21-12Ol-OO13, School of A v i a t i o n Med., J u n e , 1954. • J. KRUH, Bull. soc. chim. biol., 34 (1952) 778. s T. H. J. HUISMAN AND ANDR~E DOZY, Biochim. Biophys. Acta, 2o (1956) 4o0. 0 p. C. VAN DER SCHAAF AND T. H. J. HUISMAN, Biochim. Biophys. Acta, 17 (I955) 81. 1o K. BETKE AND I. GREINACHER, Acta Haematol., I1 (I954) 378. Received April 5th, I956
SHORT COMMUNICATIONS
VOL. 2 0 (1956)
Some observations about the heterogeneity of haemoglobin in aluminum oxide chromatography The heterogeneity of normal adult haemoglobin and of some a b n o r m a l haemoglobins is reported by several workers using different methods, for instance alkali d e n a t u r a t i o n 1, salting out s, chrom a t o g r a p h y on Amberlite IRC 5 o3,4, p a p e r electrophoresis 5 and c h r o m a t o g r a p h y on AIzO36,L Using the AI,O 3 column, VAN FOSSAN8 succeeded in fractionating H b of both adult and fetus into three distinct components. As this method seemed suitable for detecting the heterogeneity of H b some experiments were carried out to check the occurrence in blood of the different comp o n e n t s postulated. The results of this research are reported here. The haemoglobins used (A, F, 13 and C) have been described elsewhere 8. The preparation was carried out as reported earlierg; b u t the haemoglobin solutions (containing xo g per cent of COHb) were never dialysed, except in a few special cases described below. Two samples of a l u m i n i u m oxide suitable for c h r o m a t o g r a p h i c analysis were used, one obtained from Merck and one from the British Drug Houses Inc. (13.D.H.). The general procedure was similar to those described by VAN FOSSAN~: The A1203 was poured into the tube (diameter o.9 to 2. 5 cm × 5 to 2o cm) as a thick aqueous suspension and allowed to settle. Small a m o u n t s of C O H b (5-5 ° mg) were p u t on the column and development was carried out with distilled water. The p H of the eluate fractions collected varied between 9.5 and i o . I . Two red water-mobile bands descended in the column close to each other, while a third one which was immobile at the top was eluted with o.35 % ammonia. The a m o u n t of H b in each fraction was determined with a Beckman s p e c t r o p h o t o m e t e r at 5700 A. The results of the fractionation of various h u m a n haemoglobins (A, I3, C and H b from cord blood) are given in Table I. Since a relatively large deviation exists for the a m o u n t s of the fractions I and II, it will be clear t h a t no significant differences exist between the four haemoglobins studied. I t will be noted t h a t the ratio between the three fractions is quite different from t h a t reported by VAN FOSSAN, w h o found average values of 4 8 % (I), 3°/0 (II) and 49% ( I I I ) for adult Hb. These differences m a y be explained by differences in the batches of AlzO v though when B.D.H. and Merck aluminium oxide was used a p p r o x i m a t e l y similar results were obtained (Table I). The influence of dialysis on the H b solutions is remarkable. W h e n the COHb solution was dialysed against distilled water at 4 ° C for 96 hours the a m o u n t of the ammonia-mobile fraction decreased from 19% to 8 % , while moreover only one water-mobile fraction was observed (Table I). The effect of dialysing the H b solutions against 0.35 % a m m o n i a (45 hours at 4 ° C and 3 h o u r s against distilled water in order-to remove the ammonia) is still greater: only 5 % of the total a m o u n t of h a e m o g l o b i n remained at the top of the column (Table I). In some experim e n t s the dialysate was concentrated by freeze-drying and recombined in a threefold equivalent a m o u n t with the dialysed H b solution. The chromatographic separation showed an increase in the a m o u n t of the a m m o n i a - m o b i l e fraction (from 5% before to 2I % after the recombination), TABLE I CHROMATOGRAPHY
A llO, used
Merck BDH BDH 13DH BDH BDH BDH BDH 13DH BDH
OF
HAEMOGLOBIN
ON
Pr etreatrnent
---
---96 h dialysis against distilled water 8 h dialysis against o.35 % N H 3 45 h dialysis against 0.35 % N H 3 45 h dialysis against 0.35 % N H v recombined with a threefold equivalent a m o u n t of the dialysate 45 h dialysis against 0.35 % NH3, recombined with a threefold equimolar a m o u n t of G S H
AIaO a UNDER
Hb used
A A B C F A A A A A
VARYING
CONDITIONS
.Pem~,¢agt o! tke diOtrent ]raaions I
II
III
25 35 ± 5 (n = 8) 33 27 3~
52 46 :t: 5 (n = 8) 36 5z 42 92 85 95 79
23 19 ± 2 (~ = s) 31 2I 27 8 15 5 2i
8i
I9
voL. 20 (1956)
SHOAT COMMUNICATIONS
571
while in s o m e e x p e r i m e n t s two water-mobile fractions were observed. As g l u t a t h i o n e (GSH) is t h e m a i n c o m p o n e n t in t h e dialysate, similar e x p e r i m e n t s were carried o u t b y r e c o m b i n i n g t h e dialysed H b solution w i t h a threefold e q u i m o l a r a m o u n t of GSH. T h e r e s u l t s are q u i t e similar to those o b t a i n e d with t h e c o n c e n t r a t e d di70 a l y s a t e (Table I). F r o m t h e s e d a t a it m a y be c o n c l u d e d t h a t G S H is a p r o m i n e n t factor in t h e resolution of h a e m o g l o b i n into t h r e e c o m p o n e n t s . H o w e v e r similar results were o b t a i n e d on e x a m i n a t i o n of h a e m o g l o b i n solutions with ._gso different p H ' s (obtained b y slowly a d d i n g v e r y small a m o u n t s of hydrochloric acid). T h e d a t a of Fig. i show t h a t a decrease of t h e p H from 8.8 to 6.0 g a v e a two- to threefold increase in o t h e a m o u n t of t h e a m m o n i a - m o b i l e fraction. E This increase is still g r e a t e r w h e n c o n c e n t r a t e d "r1,0o dialysate or G S H was also a d d e d (Fig. i). z P r e t r e a t m e n t of t h e a l u m i n u m oxide with distilled w a t e r e x e r t e d a g r e a t influence on t h e p e r c e n t a g e of t h e a m m o n i a - m o b i l e fraction. I n s o m e e x p e r i m e n t s s m a l l c o l u m n s (0.9 × 5.0 cm) filled with AltO s were w a s h e d with large a m o u n t s of distilled w a t e r a n d t h e n used for c h r o m a t o g r a p h i c analyses. It w a s found t h a t t h e total q u a n t i t y of h a e m o g l o b i n , I 1 r e m a i n e d a t t h e t o p of t h e c o l u m n after a 6 7 8 pH p r e t r e a t m e n t With 15oo to 3ooo ml of distilled Fig. 1. T h e influence of t h e p H . o f t h e H b solution water. I n t h o s e cases t h e p H of t h e e l u a n t on t h e percentage of t h e a m m o n i a - m o b i l e fraction. was decreased f r o m 9.5 to 7.2 to 6.8. • • without addition; O O recombined F r o m t h e s e e x p e r i m e n t s it will be clear w i t h t h e c o n c e n t r a t e d dialysate; × x recomt h a t t h e s e p a r a t i o n of h a e m o g l o b i n into t h r e e bined w i t h GSH. I n all cases a d j u s t m e n t of t h e c o m p o n e n t s b y a l u m i n i u m oxide c h r o m a t o p H w i t h HCL g r a p h y is d e p e n d e n t on different e x p e r i m e n t a l conditions. I t s e e m s v e r y e a s y to c o n v e r t one c o m p o n e n t into t h e o t h e r b y slight e x p e r i m e n t a l alterations. T h e effect of small a m o u n t s of hydrochloric acid a n d of g l u t a t h i o n e in increasing t h e p e r c e n t a g e of t h e a m m o n i a - m o b i l e fraction m a y be explained b y a s s u m i n g t h a t s m a l l a m o u n t s of AltO * are c o n v e r t e d into AI(OH)s. This a l u m i n i u m h y d r o x i d e is able to absorb large a m o u n t s of haemoglobin TM. T h e s a m e e x p l a n a t i o n m a y be valid for t h e striking effect of w a s h i n g t h e A l t O s c o l u m n w i t h distilled w a t e r ; it m a y be t h a t larger a m o u n t s of AlsO a are c o n v e r t e d into AI(OH)a, t h u s a c c o u n t i n g for t h e high c a p a c i t y of s u c h a c o l u m n for a b s o r b i n g h a e m o g l o b i n . W e believe, therefore, t h a t t h e a p p a r e n t h e t e r o g e n e i t y of h a e m o g l o b i n as s h o w n b y a l u m i n i u m oxide c h r o m a t o g r a p h y is d u e to e x p e r i m e n t a l c o n d i t i o n s ; t h e use of this m e t h o d for p r o v i n g h a e m o g l o b i n h e t e r o g e n e i t y is strictly limited. H. K. PRINS Department o/Pediatrics, University o/Groningen (The Netherlands) T . H . J . HUZSMAN 1 R . BRXNKMA~, A. WILDSCHUT AND A. WITTERMAN, J. Physiol., 80 (1934) 377. t j . ROCHE AND Y. DERRIEN, Le Sang, 24 (1953) 97. • M. MORRISON AND J. CooK, Science, 12o (1955) 920. t H. K. PRINS AND T. H. J. HUISMAN, Nature (in t h e press). s H. G. KUNKEL AND G. WALLENIUS, Scie~,ce, 122 (I955) 288. • D. D. VAN FOSSAN, Federation Prec., 13 (1954) 156; R e p o r t No. 7; P r o j e c t No. 21-12Ol-OO13, School of A v i a t i o n Med., J u n e , 1954. • J. KRUH, Bull. soc. chim. biol., 34 (1952) 778. s T. H. J. HUISMAN AND ANDR~E DOZY, Biochim. Biophys. Acta, 2o (1956) 4o0. 0 p. C. VAN DER SCHAAF AND T. H. J. HUISMAN, Biochim. Biophys. Acta, 17 (I955) 81. 1o K. BETKE AND I. GREINACHER, Acta Haematol., I1 (I954) 378. Received April 5th, I956