19
Clinica Chimica Acta, 90 (1978) 0 Elsevier/North-Holland
19-28 Biomedical Press
CCA 9605
THIN-LAYER ISOELECTRIC FOCUSING OF HEMOGLOBIN VARIANTS : SCREENING AND DETERMINATION OF ISOELECTRIC POINTS
A. GIULIANI,
Laboratorio (Received
M. MARINUCCI,
M.P. CAPPABIANCA,
D. MAFFI
and L. TENTORI
*
di Patologia non Infettiva, Instituto Superiore di Sanitci, Rome (Italy)
March
28th,
1978)
Summary The high resolving power of thin-layer isoelectric focusing was applied for screening some hemoglobin variants classified on the basis of their electrophoretie mobility in: electrophoretically slow variants (as Hb A,), electrophoretically slow variants (as Hb S), electrophoretically fast variants (Hbs type J). An analysis of the variant compounds has been performed, and the corresponding pl values were determined in whole hemolysate.
Introduction In the last few years isoelectric focusing (IEF) has been introduced as a more selective method than electrophoresis (EF) for preparative and analytical purposes in biochemical field. Its resolving power [l--11] often enables the detection of heterogeneity on electrophoretically homogeneous specimens. This technique was applied to study different biological molecules : glycoproteins, lipoproteins, hormones, enzymes, metalloproteins, as well as to investigate immunoglobulins and cellular membrane constituents. Recently IEF was applied for identifying and mapping many hemoglobin variants [ 12-181. The aim of the present work is the detection of differences between variants, electrophoretically unresolved, by the greater resolving power of IEF. Therefore we paid special attention to different patterns of electrophoretically simi* Correspondence should be addressed to: Professor L. Tentori, Laboratorio di Patologia non Infettiva, Institute Superiore de Sanitl, vi& Regina Elena, 299-00161 Roma. Italy.
20
lar variants, and to observing whether IEF could provide an answer to functional behavior of the modified molecule. The precision of measuring the pl values of the samples on the gel has been also given. The variants were divided as follows: electrophoretically slow variants as Hb Al, electrophoretically slow variants as Hb S, electrophoretically fast variants : Hbs type J. The IEF behaviour of these was established and their pl values were determined. Materials and methods Reagents
Ampholine@ LKB 40% w/v solutions in pH ranges 5--8/8-9.5 or 6-B/7-9 in order to obtain a gradient of 5.5-9 or 6.5-8.5. The use of two different mixtures of carriers did not modify mobility or resolving power. N&V’-Methylene bisacrylamide, acrylamide, sucrose and riboflavin, were all produced by the British Drug Houses (BDH) Ltd., London, U.K. Preparation
of the hemoglobins
Some of the hemoglobins reached our laboratory as HiCN hemolysates. Other samples as whole blood; in this case they were lysed by addition of distilled water and carbon tetrachloride. The hemolysates were diluted with distilled water (1 : 3) and 0.05 ml of 2% w/v KCN was added. All the samples were stored at 4°C as HiCN (15 ~1 KCN 2%/ml Hb) and used within three months, without any modification of IEF patterns. All samples were submitted to preliminary electrophoresis on cellulose acetate strips (Titan III, Helena) in Tris-EDTA-Borate buffer pH 8.4/0.025 I. Gel preparation
The gel was prepared as follows: 10 ml 29% w/v acrylamide. 10 ml 0.9% w/v methylene bisacrylamide. 36 ml 20.8% w/v sucrose. 1.5 ml Ampholine@ of the pH range 5-8 or 6-S. 1.5 ml Ampholine@ of the pH range 8-9.5 or 7-9. The stock solutions of acrylamide and methylene bisacrylamide were used during two weeks. The sucrose solution was freshly prepared. The solutions were thoroughly mixed and deaerated for about two minutes under vacuum. After deareation 0.6 ml of 0.004% w/v riboflavin was added to the mixture. Preparation
of the slides and IEF
The plates were prepared according the method of Awdeh et al. [6] modified by Vesterberg [7]. For the IEF, the Multiphor 2117 LKB system was used. Complete polymerization was done with fluorescent light, within not less than 2.5 h, the gels were stored overnight at 4°C before focusing. Pieces of Whatman 3 MM paper 0.3 X 0.7 cm (7-8 PI), or 0.5 X 1 cm (12-
21
15 ~1) were soaked in sample solution and placed at about 2 cm from the anode and the cathode. The anodical and the cathodical strips were soaked in 0.5 M H,SO, and NaOH solutions, respectively. Focusing
procedure
Initial power was of 8-10 watts. Within 90 min the voltage was gradually increased from 250 V to 1000 V, and maintained constant during the whole experiment (3 h). The experimental temperature was 10°C. Other conditions were reported previously (1978, LAB, in press). Finally, the gels were placed for 30 min in fixing solution. Experimental
results
Electrophoretically
slow variants as Hb A2
Fig. 1 shows the IEF patterns. The pl values are reported in Table I. The 0 Padova, 0 Indonesia Hbs (Fig. 2) and Hb A*, though resolved, have p1 values very close to each other and to Hb A*. Hb C is separated from the Hb AZ [12,17,19]; furthermore the two fractions exhibit a difference in pl. Electrophoretically
slow variants as Hb 5’ (Fig. 3, Table II)
Sometimes their detection is very difficult, especially for Hb Lepore-Boston [20] and for Hb A in the Hb G Ferrara hemolysate (Fig. 4) because of the relative low amounts of fraction.
Fig. 1. ElectrophoreticaIIy slow variants as Hb AZ. 1. Hb 0 Indonesia; 2, Hb C + Hb S; 3. Hb C: 4. Hb A2 5, Hb Normal: 6, Hb 0 Indonesia: 7, Hb 0 Padova.
;
22
TABLE
I
NalTle
Substitution
No.
of
S.D.pI
PI
S.D.AHbA
*HbA
trials A2
-
C 0
/36 Glu -+ LYS Indonesia
0 Padova
11
7.35
to.06
9
7.47
to.06
0.44
fO.06
-
01116
Glu -+ Lys
14
7.34
to.07
0.37
r0.03
a30
Glu -+ Lys
15
7.33
to.07
0.39
to.06
In Hasharon and Q India Hbs, the same amino-acidic substitution (Asp + His) of an external residue, though in a different position, slightly changes the pI (Fig. 5). Electrophoretically
fast variants as Hbs type J (Fig. 6, Table III)
All the examined hemoglobins have an electrophoretic fast mobility. The J. Norfolk [21] and J. Oxford Hbs, having the same amino-acidic substitution (Gly -+ Asp) in alpha chain, show very similar mobility in IEF (Fig. 7). The Lufkin and Baltimore Hbs, having the substitution Gly + Asp in beta chain, in IEF show a different behavior: i.e. the pI of Hb Baltimore is acid, and the hemolysate shows a band more acid than Hb A, as it is detectable in a mixture Hb Normal + Hb Baltimore (Fig. 8). As expected, the same substitution (Gly + Asp) on different chains gives different pI values. The alpha substitution of the J Rovigo and J Paris Hbs causes different behavior on IEF (Table III, Fig. 9). The 8-10 samples of Fig. 6 show modification of the Hb J Rovigo pattern with respect to Fig. 9, and this con-
1 Fig.
2
2. ElectrophoreticaIly
4,HbA2.
3 slow
variants
4 as Hb
AZ.
1. Hb
0
Indonesia:
2. Hb
0
Padova;
3, Hb
0
Indonesia;
Fig. 3. Electropboretically slow variants as Hb S. 1, Hb F + Hb A; 2, Hb Waco: 3, Hb S Travis; 4, Hb Hasharon; 5, Hb Philadelphia; 6, Hb Russ; 7, Hb Kempsey; 8, Hb G San Jose (beterozygous); 9, Hb G San Jose (double heterozygous); 10, Hb Lepore Boston; 11. Hb Gavello: 12, Hb G Ferrara; 13, Hb Punjab; 14, Hb Q India; 15, Hb C + Hb S; 16, Hb A + Hb S; 17, Hb Nomdl.
24
TABLE
II Substitution
No.of
PI
S.D.pI
AHbA
S.D.AHbA
trials
S
VaI
4
7.21
to.06
0.25
to.02
Glu -+a Gln
3
7.19
kO.09
0.21
r0.02
057
Am-+
LYS
6
7.32
kO.09
0.29
kO.03
p47
Asp+
Gly
8
7.26
+0.05
0.25
to.04
687
Glu
p116
His
5
7.14
to.09
0.17
r0.04
06 Glu -+
D Punjab
0121
G Ferrari GaVelI Lepore
Boston
G San
Jo&
G San Jose Kempsey Russ
*
p7 Glu +
Gly
7
7.15
to.07
0.14
to.04
* *
/37 Glu -+ Gly
6
7.13
kO.08
0.15
to.02
4
7.11
io.07
0.10
r0.01
8
7.17
to.05
0.18
to.01
* **
***
099
ASP?,
a51
Gly
Asn
-+ Am
G Philadelphia
a68
Am-t
LYS
4
7.17
to.05
0.19
_+O.Ol
Hasharon
a47
Asp+
His
14
7.29
r0.06
0.26
to.02
0164 Asp+.
His
6
7.22
kO.07
0.23
to.02
040
LYS
5
7.05
to.06
0.05
to.01
4
7.14
to.07
0.23
to.02
6
7.23
20.06
0.23
to.02
Q India Waco
***
***
Arg +
F S Travis
* ** ***
***
Heterozygous. Double Variants.
heterozygous Kindly
Hb
provided
G San Jo&/P-thal. by
Professor
R.M.
Schmidt
(Atlanta).
firms the instability of the variant. Furthermore the Hb J Rovigo shows a pl 7.30, quite near to that calculated for HbAz. The J Sardegna and J Mexico Hbs (Fig. 6) on which there is a base-acid substitution in an alpha chain, do not present significant pl variations.
Fig.
4.
1, Hb
Fig.
5. 1, Hb
Lepore Hasharon;
Boston: 2, Hb
2, Hb
G Ferrara.
Q India.
25
26 TABLE III NEUlE
Substitution
J Norfolk J Oxford J Rovigo J Paris 3 Mexico J Sardegna JLufkin* J Baltimore
cr57 GIY -+ Asp 015 Gly -+ ASP a53 Ala -+ Asp ~112 Ala + Asp n54 Gin -+ Glu a50 His +ASP @29 Gly *Asp P16 Gly + Asp
No. of triats 6.79 6.81 6.97 6.77 6.85 6.73 6.86 6.74
S.D.pi
AHbA
S.D-AHbA
to.07 r0.06 to.06 -to.03 +0.06 to.05 tO.06 20.07
0.21
to.02 to.01 rO.O1 +0.04 10.04 LtO.05 to.05 to.02
0.19 0.10 0.20 0.14 0.26 0.13 0.20
* Variants. Kindly provided by Professor R.M. Schmidt (Atlanta).
Fig. 7. 1, Hb J Oxford; 2, Hb J Norfolk. Fig. 8. 1, Hb J Baltimore f Hb Normal; 2, Hb Normal; 3, Hb J Baltimore; 4, Hb J Lufkin. Fig. 9. 1, Hb J Rovigo; 2, Hb J Paris.
Discussion The results demonstrate that our prelimin~ aims have been realised; the analysis of IEF patterns of variant hemolysate can provide answers on the structure of modified hemoglobins, because this method, to a greater extent than electrophoresis, can reveal either the nature or the position of the substitution. Theoretically, if two variants have the same substitution, though in different position, the pK value of amino acid, and therefore the pf of protein, should not undergo a si~ific~t change: on the contrary, the position of sub-
27
stitution and the presence in the molecule of different ionizable groups modify the actual pK value of the amino acid, giving rise to different pl values of the two variants. Usually, an external substitution leads to greater modification of the pl: i.e. in J Lufkin and J Baltimore Hbs, there is the same Gly + Asp substitution but in Hb J Lufkin the substitution (/3 29) is internal, in Hb J Baltimore is external (/3 16): according to that, pl of Hb J Baltimore is more anodic than Hb J Lufkin, with respect to Hb A (~10.92 -I 0.03,1978, LAB, in press). IEF points out the different p1 values in spite of a substitution of same nature, owing to modified interaction of the neighboring charged groups: e.g., though a similar substitution, the Hb J Sardegna (0150 His + Asp) has a p1 more anodic than Hb J Mexico (a 54 Gln -+ Glu), because of more incisive positional effect of the substitution. Indeed in normal hemoglobin His (01 50) lies in CD 8, a non-helical segment, and twists a salt bridge with Glu (a 30) of the same chain; this bridge is lost in the Asp ((Y50) of Hb J Sardegna. In Hb H Mexico, Glu (a 54) takes place in helical segment E 3, which, even though near to heme, does not seem to interfere with it in modifying the tertiary structure of the molecule. It follows that IEF can provide a useful means for understanding the tertiary structure of proteins. The use of pl detection directly on the slab shows a wide standard deviation (S.D.,,) which depends upon the surface dimensions of the reading electrode also when the influence of the periods of experimental, the polymerization of the gel, the unexpected splittings of temperature or power were removed and the shape and stability of the gradients were achieved. The absolute value of the difference of pH (ApH) between variant and Hb A fractions represents a significant value, because this is more steady (Tables I, II, III). It may be useful to carry out IEF of whole hemolysate for emphasizing minor components, such as Hb A3 or hybrids between the variant chain and the normal one [ 13,221. In conclusion, we can confirm the greater resolving power of IEF related to cellulose acetate EF for most of the variants having the same electrophoretic mobility; furthermore, the method is fast and easy since it requires neither particular handling of the sample nor a definite quantity for it; it is possible to compare a lot of samples together on the same slab; so it is useful simultaneously with electrophoresis in routine screening. The precision in the reading of pl values may be improved with a needlepoint electrode, or by using a densitometric method, in order to obtain standard patterns as has already been carried out in human serum EF [ 231. Acknowledgements We thank Mr. Giovanni
Franc0 for his excellent
technical
assistance.
References 1
Fawcett,
2
Dwsdale,
J.S. J.W.,
(1968)
FEBS
Righetti.
Lett.
1. 80-82
P. and Bunn.
H.F.
(1971)
Biochim.
Biophys.
Acta
229.42-50
28 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 2?. 22 23
Bunn. H.F. and DrysdaIe. J.W. (1971) Biochim. Biophys. Acta 229, 51-57 Miles, L.E.M. and Simmons, J.E. (1972) Anal. Biochem. 49.109-117 Mslik, N. and Ben%, A. (1972) Anal. Biochem. 49.173-176 Awdeh, Z.L.. WiIIiamson. A.R. and Askonas, B.A. (1968) Nature 219, 66-67 Vesterberg, 0. (1972) Biochim. Biophys. Acta 257, 11-19 Beeley, J.A., Stevenson, S.M. and Beeley. J.G. (1972) Biochim. Biophys. Acta 285,293-300 Sijderholm, J.. Allestam, P. and Wadstriim, T. (1972) FEBS Lett. 24, 89-92 Aburthnott. J.F. and Beeley, J.A. (1975) in Isoelectric Focusing, Butterworths, London Righetti. P.G. and DrysdaIe. J.W. (1976) in Laboratory Techniques in Biochemistry and Molecular Biology (Work. T.S. and Work, E., eds.), Vol. 5. Part II: Isoelectric Focusing, North-Holland Publishing Company, Amsterdam DrysdaIe. J.W.. Righetti, P.G. and Bunn, H.F. (1971) Biochim. Biophys. Acta 229,42-50 Bunn. H.F. (1973) Ann. N.Y. Acad. Sci. 209, 345-353 Bunn, H.F. and Drysdale. J.W. (1971) Biochim. Biophys. Acta 229, 51-57 Jeppsson, J.O. and Berglund, S. (1972) CU. Chim. Acta 40.153-158 Schneider, R.G. (1974) CIin. Chem. 20,1111-1115 Koepke. J.A., Thoma. J.F. and Schmidt, R.M. (1975) Clin. Chem. 21.1953-1955 Monte, M.. Beauzard, Y. and Rosa, J. (1976) Am. J. Clin. Pathol. 66. 753-769 Krishnamoorthy, R., Wajcman. H. and Labie, D. (1976) Clin. Chim. Acta 69. 203-209 BagIioni. C. (1965) Biochim. Biophys. Acta 97, 37-46 Lorkin. P.A., Huntsman, R.G., Ager, J.A.M., Lehmann, H., VeIIa, F. and Darbre. P.D. (1975) Biochim. Biophys. Acta 379.22-27 Bunn, H.F. and McDonough, M. (1974) Biochemistry 13.988-993 Badiali. M.. Ballard, H.D., Bertuzzi, A., Matteuchi, M. (1975) Comunication to Nato-Asi Summer School on Pattern Recognition Theory and Application, Ile de Bendor (France)
Clinica Chimica Acta, 90 (1978) 0 Elsevier/North-Holland
19-28 Biomedical Press
CCA 9605
THIN-LAYER ISOELECTRIC FOCUSING OF HEMOGLOBIN VARIANTS : SCREENING AND DETERMINATION OF ISOELECTRIC POINTS
A. GIULIANI,
Laboratorio (Received
M. MARINUCCI,
M.P. CAPPABIANCA,
D. MAFFI
and L. TENTORI
*
di Patologia non Infettiva, Instituto Superiore di Sanitci, Rome (Italy)
March
28th,
1978)
Summary The high resolving power of thin-layer isoelectric focusing was applied for screening some hemoglobin variants classified on the basis of their electrophoretie mobility in: electrophoretically slow variants (as Hb A,), electrophoretically slow variants (as Hb S), electrophoretically fast variants (Hbs type J). An analysis of the variant compounds has been performed, and the corresponding pl values were determined in whole hemolysate.
Introduction In the last few years isoelectric focusing (IEF) has been introduced as a more selective method than electrophoresis (EF) for preparative and analytical purposes in biochemical field. Its resolving power [l--11] often enables the detection of heterogeneity on electrophoretically homogeneous specimens. This technique was applied to study different biological molecules : glycoproteins, lipoproteins, hormones, enzymes, metalloproteins, as well as to investigate immunoglobulins and cellular membrane constituents. Recently IEF was applied for identifying and mapping many hemoglobin variants [ 12-181. The aim of the present work is the detection of differences between variants, electrophoretically unresolved, by the greater resolving power of IEF. Therefore we paid special attention to different patterns of electrophoretically simi* Correspondence should be addressed to: Professor L. Tentori, Laboratorio di Patologia non Infettiva, Institute Superiore de Sanitl, vi& Regina Elena, 299-00161 Roma. Italy.
20
lar variants, and to observing whether IEF could provide an answer to functional behavior of the modified molecule. The precision of measuring the pl values of the samples on the gel has been also given. The variants were divided as follows: electrophoretically slow variants as Hb Al, electrophoretically slow variants as Hb S, electrophoretically fast variants : Hbs type J. The IEF behaviour of these was established and their pl values were determined. Materials and methods Reagents
Ampholine@ LKB 40% w/v solutions in pH ranges 5--8/8-9.5 or 6-B/7-9 in order to obtain a gradient of 5.5-9 or 6.5-8.5. The use of two different mixtures of carriers did not modify mobility or resolving power. N&V’-Methylene bisacrylamide, acrylamide, sucrose and riboflavin, were all produced by the British Drug Houses (BDH) Ltd., London, U.K. Preparation
of the hemoglobins
Some of the hemoglobins reached our laboratory as HiCN hemolysates. Other samples as whole blood; in this case they were lysed by addition of distilled water and carbon tetrachloride. The hemolysates were diluted with distilled water (1 : 3) and 0.05 ml of 2% w/v KCN was added. All the samples were stored at 4°C as HiCN (15 ~1 KCN 2%/ml Hb) and used within three months, without any modification of IEF patterns. All samples were submitted to preliminary electrophoresis on cellulose acetate strips (Titan III, Helena) in Tris-EDTA-Borate buffer pH 8.4/0.025 I. Gel preparation
The gel was prepared as follows: 10 ml 29% w/v acrylamide. 10 ml 0.9% w/v methylene bisacrylamide. 36 ml 20.8% w/v sucrose. 1.5 ml Ampholine@ of the pH range 5-8 or 6-S. 1.5 ml Ampholine@ of the pH range 8-9.5 or 7-9. The stock solutions of acrylamide and methylene bisacrylamide were used during two weeks. The sucrose solution was freshly prepared. The solutions were thoroughly mixed and deaerated for about two minutes under vacuum. After deareation 0.6 ml of 0.004% w/v riboflavin was added to the mixture. Preparation
of the slides and IEF
The plates were prepared according the method of Awdeh et al. [6] modified by Vesterberg [7]. For the IEF, the Multiphor 2117 LKB system was used. Complete polymerization was done with fluorescent light, within not less than 2.5 h, the gels were stored overnight at 4°C before focusing. Pieces of Whatman 3 MM paper 0.3 X 0.7 cm (7-8 PI), or 0.5 X 1 cm (12-
21
15 ~1) were soaked in sample solution and placed at about 2 cm from the anode and the cathode. The anodical and the cathodical strips were soaked in 0.5 M H,SO, and NaOH solutions, respectively. Focusing
procedure
Initial power was of 8-10 watts. Within 90 min the voltage was gradually increased from 250 V to 1000 V, and maintained constant during the whole experiment (3 h). The experimental temperature was 10°C. Other conditions were reported previously (1978, LAB, in press). Finally, the gels were placed for 30 min in fixing solution. Experimental
results
Electrophoretically
slow variants as Hb A2
Fig. 1 shows the IEF patterns. The pl values are reported in Table I. The 0 Padova, 0 Indonesia Hbs (Fig. 2) and Hb A*, though resolved, have p1 values very close to each other and to Hb A*. Hb C is separated from the Hb AZ [12,17,19]; furthermore the two fractions exhibit a difference in pl. Electrophoretically
slow variants as Hb 5’ (Fig. 3, Table II)
Sometimes their detection is very difficult, especially for Hb Lepore-Boston [20] and for Hb A in the Hb G Ferrara hemolysate (Fig. 4) because of the relative low amounts of fraction.
Fig. 1. ElectrophoreticaIIy slow variants as Hb AZ. 1. Hb 0 Indonesia; 2, Hb C + Hb S; 3. Hb C: 4. Hb A2 5, Hb Normal: 6, Hb 0 Indonesia: 7, Hb 0 Padova.
;
22
TABLE
I
NalTle
Substitution
No.
of
S.D.pI
PI
S.D.AHbA
*HbA
trials A2
-
C 0
/36 Glu -+ LYS Indonesia
0 Padova
11
7.35
to.06
9
7.47
to.06
0.44
fO.06
-
01116
Glu -+ Lys
14
7.34
to.07
0.37
r0.03
a30
Glu -+ Lys
15
7.33
to.07
0.39
to.06
In Hasharon and Q India Hbs, the same amino-acidic substitution (Asp + His) of an external residue, though in a different position, slightly changes the pI (Fig. 5). Electrophoretically
fast variants as Hbs type J (Fig. 6, Table III)
All the examined hemoglobins have an electrophoretic fast mobility. The J. Norfolk [21] and J. Oxford Hbs, having the same amino-acidic substitution (Gly -+ Asp) in alpha chain, show very similar mobility in IEF (Fig. 7). The Lufkin and Baltimore Hbs, having the substitution Gly + Asp in beta chain, in IEF show a different behavior: i.e. the pI of Hb Baltimore is acid, and the hemolysate shows a band more acid than Hb A, as it is detectable in a mixture Hb Normal + Hb Baltimore (Fig. 8). As expected, the same substitution (Gly + Asp) on different chains gives different pI values. The alpha substitution of the J Rovigo and J Paris Hbs causes different behavior on IEF (Table III, Fig. 9). The 8-10 samples of Fig. 6 show modification of the Hb J Rovigo pattern with respect to Fig. 9, and this con-
1 Fig.
2
2. ElectrophoreticaIly
4,HbA2.
3 slow
variants
4 as Hb
AZ.
1. Hb
0
Indonesia:
2. Hb
0
Padova;
3, Hb
0
Indonesia;
Fig. 3. Electropboretically slow variants as Hb S. 1, Hb F + Hb A; 2, Hb Waco: 3, Hb S Travis; 4, Hb Hasharon; 5, Hb Philadelphia; 6, Hb Russ; 7, Hb Kempsey; 8, Hb G San Jose (beterozygous); 9, Hb G San Jose (double heterozygous); 10, Hb Lepore Boston; 11. Hb Gavello: 12, Hb G Ferrara; 13, Hb Punjab; 14, Hb Q India; 15, Hb C + Hb S; 16, Hb A + Hb S; 17, Hb Nomdl.
24
TABLE
II Substitution
No.of
PI
S.D.pI
AHbA
S.D.AHbA
trials
S
VaI
4
7.21
to.06
0.25
to.02
Glu -+a Gln
3
7.19
kO.09
0.21
r0.02
057
Am-+
LYS
6
7.32
kO.09
0.29
kO.03
p47
Asp+
Gly
8
7.26
+0.05
0.25
to.04
687
Glu
p116
His
5
7.14
to.09
0.17
r0.04
06 Glu -+
D Punjab
0121
G Ferrari GaVelI Lepore
Boston
G San
Jo&
G San Jose Kempsey Russ
*
p7 Glu +
Gly
7
7.15
to.07
0.14
to.04
* *
/37 Glu -+ Gly
6
7.13
kO.08
0.15
to.02
4
7.11
io.07
0.10
r0.01
8
7.17
to.05
0.18
to.01
* **
***
099
ASP?,
a51
Gly
Asn
-+ Am
G Philadelphia
a68
Am-t
LYS
4
7.17
to.05
0.19
_+O.Ol
Hasharon
a47
Asp+
His
14
7.29
r0.06
0.26
to.02
0164 Asp+.
His
6
7.22
kO.07
0.23
to.02
040
LYS
5
7.05
to.06
0.05
to.01
4
7.14
to.07
0.23
to.02
6
7.23
20.06
0.23
to.02
Q India Waco
***
***
Arg +
F S Travis
* ** ***
***
Heterozygous. Double Variants.
heterozygous Kindly
Hb
provided
G San Jo&/P-thal. by
Professor
R.M.
Schmidt
(Atlanta).
firms the instability of the variant. Furthermore the Hb J Rovigo shows a pl 7.30, quite near to that calculated for HbAz. The J Sardegna and J Mexico Hbs (Fig. 6) on which there is a base-acid substitution in an alpha chain, do not present significant pl variations.
Fig.
4.
1, Hb
Fig.
5. 1, Hb
Lepore Hasharon;
Boston: 2, Hb
2, Hb
G Ferrara.
Q India.
25
26 TABLE III NEUlE
Substitution
J Norfolk J Oxford J Rovigo J Paris 3 Mexico J Sardegna JLufkin* J Baltimore
cr57 GIY -+ Asp 015 Gly -+ ASP a53 Ala -+ Asp ~112 Ala + Asp n54 Gin -+ Glu a50 His +ASP @29 Gly *Asp P16 Gly + Asp
No. of triats 6.79 6.81 6.97 6.77 6.85 6.73 6.86 6.74
S.D.pi
AHbA
S.D-AHbA
to.07 r0.06 to.06 -to.03 +0.06 to.05 tO.06 20.07
0.21
to.02 to.01 rO.O1 +0.04 10.04 LtO.05 to.05 to.02
0.19 0.10 0.20 0.14 0.26 0.13 0.20
* Variants. Kindly provided by Professor R.M. Schmidt (Atlanta).
Fig. 7. 1, Hb J Oxford; 2, Hb J Norfolk. Fig. 8. 1, Hb J Baltimore f Hb Normal; 2, Hb Normal; 3, Hb J Baltimore; 4, Hb J Lufkin. Fig. 9. 1, Hb J Rovigo; 2, Hb J Paris.
Discussion The results demonstrate that our prelimin~ aims have been realised; the analysis of IEF patterns of variant hemolysate can provide answers on the structure of modified hemoglobins, because this method, to a greater extent than electrophoresis, can reveal either the nature or the position of the substitution. Theoretically, if two variants have the same substitution, though in different position, the pK value of amino acid, and therefore the pf of protein, should not undergo a si~ific~t change: on the contrary, the position of sub-
27
stitution and the presence in the molecule of different ionizable groups modify the actual pK value of the amino acid, giving rise to different pl values of the two variants. Usually, an external substitution leads to greater modification of the pl: i.e. in J Lufkin and J Baltimore Hbs, there is the same Gly + Asp substitution but in Hb J Lufkin the substitution (/3 29) is internal, in Hb J Baltimore is external (/3 16): according to that, pl of Hb J Baltimore is more anodic than Hb J Lufkin, with respect to Hb A (~10.92 -I 0.03,1978, LAB, in press). IEF points out the different p1 values in spite of a substitution of same nature, owing to modified interaction of the neighboring charged groups: e.g., though a similar substitution, the Hb J Sardegna (0150 His + Asp) has a p1 more anodic than Hb J Mexico (a 54 Gln -+ Glu), because of more incisive positional effect of the substitution. Indeed in normal hemoglobin His (01 50) lies in CD 8, a non-helical segment, and twists a salt bridge with Glu (a 30) of the same chain; this bridge is lost in the Asp ((Y50) of Hb J Sardegna. In Hb H Mexico, Glu (a 54) takes place in helical segment E 3, which, even though near to heme, does not seem to interfere with it in modifying the tertiary structure of the molecule. It follows that IEF can provide a useful means for understanding the tertiary structure of proteins. The use of pl detection directly on the slab shows a wide standard deviation (S.D.,,) which depends upon the surface dimensions of the reading electrode also when the influence of the periods of experimental, the polymerization of the gel, the unexpected splittings of temperature or power were removed and the shape and stability of the gradients were achieved. The absolute value of the difference of pH (ApH) between variant and Hb A fractions represents a significant value, because this is more steady (Tables I, II, III). It may be useful to carry out IEF of whole hemolysate for emphasizing minor components, such as Hb A3 or hybrids between the variant chain and the normal one [ 13,221. In conclusion, we can confirm the greater resolving power of IEF related to cellulose acetate EF for most of the variants having the same electrophoretic mobility; furthermore, the method is fast and easy since it requires neither particular handling of the sample nor a definite quantity for it; it is possible to compare a lot of samples together on the same slab; so it is useful simultaneously with electrophoresis in routine screening. The precision in the reading of pl values may be improved with a needlepoint electrode, or by using a densitometric method, in order to obtain standard patterns as has already been carried out in human serum EF [ 231. Acknowledgements We thank Mr. Giovanni
Franc0 for his excellent
technical
assistance.
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