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Extinction coefficients for use in equations for the spectrophotometric analysis of haemoglobin mixtures
ANALYTICAL
69, 43-48 ( 1975)
BIOCHEMISTRY
Extinction
Coefficients
Spectrophotometric
for
Analysis
Use in Equations
for
the
of Haemoglobin
Mixtures
0. W. VAN ASSENDELFT AND W. G. ZIJLSTRA Laboratory
ofChernica1
Physiology,
Bloernsingel
10, Groningen,
The Netherlands
Received January 38. 1975: accepted June 5, 1975 Values of the extinction coefficients for deoxyhaemoglobin, oxyhaemoglobin. carboxyhaemoglobin, as well as those of haemiglobin between pH 6.2 and 8.8, as published by Benesch et al. (I ), are corrected. based on the internationally accepted value of I I .O for c$&.
Benesch, Benesch, and Yung ( 1) published values for molar extinction coefficients at a wavelength (h) of 540, 560, 576, and 630 nm for deoxyhaemoglobin (Hb), oxyhaemoglobin (HbO,), carboxyhaemoglobin (HbCO), and haemiglobin (ferrihaemoglobin; Hi). All haemoglobin concentrations were calculated in their study by measuring after conversion to haemiglobincyanide and using a millimolar extinction coefficient (E) at A = 540 nm (E.#$.) of 11.5. This value for E.&& had been published by Drabkin (2,3). The International Committee for Standardization in Haematology (ICSH) has, however, published recommendations for the determination of haemoglobin in human blood (4) in which a value of 1 1.0 is given for E#&. As can be seen from Table 1, where results obtained over the past 20 yr by means of various techniques are summarized, this value for E&‘& is indeed based upon abundant experimental evidence. On behalf of those wishing to use the equations given by Benesch et al. (1) for the spectrophotometric analysis of haemoglobin mixtures, the E values from their Tables 1 and 2 have been recalculated on the basis of E&;& = 1 I .O. The results are given in Tables 2 and 3. In the first column the values as published by Benesch et al. are given, in the second column these values corrected for E&$!.!. = 11.0. In the third column the values obtained by Van Assendelft (5) are included in parentheses for comparison. 43 Copyright All rights
Q 1975 by Academic Press. Inc. of reproduction in any form resewed.
Morningstar er al. (14)
Tentori et al. (13)
Wootton and Blevin (10) Van Oudheusden et nl. (11) Salvati et al. (12)
Minkowski and Swierczewski (8) Zijlstra and Van Kampen (9)
Meyer-Wilmes and Remmer (6) Remmer (7)
Author
E&
TABLE
1
Human Hb, toluene haemolysis Human whole blood Human whole blood Human Hb purified on CMC column Human Hb purified on CMC column Human whole blood Human washed cells
Material
0.03 0.07
11.02 10.97
10.99 11.06 10.95
0.05
0.05 0.08 0.03
10.68’
10.90
0.01 0.03 0.02 0.04
10.99 10.94 11.05
OR CARBON
Human Hb, toluene haemolysis
NITROGEN,
0.04 0.04 0.03 0.065 -
ON IRON,
11.0 11.0 11.09 11.19 11.15b
BASED
Horse Hb Horse Hb Human whole blood Human whole blood Foetal whole blood
AUTHORS,
s/(n)1’2
BY DIFFERENT
l&
AS REPORTED
10 6
55
10 8 46
123 35 101 14
12 12 11 4 5
n
CL,a’-dipyridyl 01, a’-dipyridyl TiCI:, ferric perchlorate
Fe; Fe: Fe; Fe;
Fe; X-ray emission spectrography
N analysis
Fe; (Y, a’-dipyridyl Fe; (Y, a’-dipyridyl Fe; a, a’-dipyridyl
o-phenanthroline TiCI, TIC!, complexon o-phenanthroline
Method Fe; Fe; Fe; Fe; Fe;
DETERMINATION’
?i u N t: L
5
B 9 Fz
>
(16)
-
n,“t
error
1 (ntot - 1)
standard
= standard
(15)
(Zi(rli
- 1)
tot
calculated
(.‘i, - k,,)‘),
= 0.01:
II = number
SF + II,
$$
of the mean:
of the mean
error
using
Total
10.88
11.00
the equation
of determinations.
Human Hb purified on CMC or Sephadex column or by dialysis against Na,-EDTA Human Hb A purified by chromatography mean
value
= 10.99:
calculated
using
the equation
C analysis
16
0.04
for E j?&
Fe; sulfosalicylic
55
0.02
acid
11~ = numwhere.r, = mean value of all determinations:\-, = mean value of the ith series of determinations; n ,,)t = total number of determinations: ber of determinations in the ith series; s, = standard deviation of the ith series of determinations; xtOf = standard deviation of all determinations. value was not published. ’ Excluded from calculation of .Y,, and s,,>, because s/(n)” the series differs more than three times the standard error of the mean from the total c Excluded from calculation of S, and stUt because mean value.
n,
&_
Total
l’.~/(h)l’L
Itano
Stigbrand
ii 2 WY
8
f
5
;1 o n
7
46
VAN
ASSENDELFT
MOLAR Deoxy A
1
540 560 570 576 630
1.08 1.34 1.16 1.01 0.115
l-Values ‘-Values A = 540 nm, 3-Values
EXTINCTION
Hb 2
1.03 1.28 1.11 0.97 0.11
AND
TABLE 2 COEFFICIENTS Oxy
3 (1.03) (1.27) (1.10) (0.98) (0.10)
1 1.53 0.906 1.23 1.65 0.015
ZIJLSTRA
(E x 10mJ)
Hb 2
1.46 0.867 1.18 1.58 0.014
given by Benesch et ~1. (1). of column 1 corrected for the millimolar equal to 11.0. given by Van Assendelft (5).
CO Hb 3 (1.43) (0.85) (1.19) (1.53) (0.02)
extinction
1 1.44 1.22 1.51 1.13 0.023
coefficient
2
3
1.38 1.17 I .44 1.08 0.022
(1.43) (1.21) (1.42) (1.12) (0.02)
of HiCN
at
When comparing the values reported by Benesch et al., after correction, with the values reported by Van Assendelft, there remain but slight differences. The values given by Van Assendelft are marginally lower in the case of HbO,; the values given by Benesch et al. are somewhat lower for HbCO. The reason for this is not quite clear. A possible explanation, however, could be found in differences in preparing the samples and possibly in differences in measuring. Benesch et al. not only washed, lysed, and centrifuged to obtain their haemoglobin solutions, but also removed the organic phosphates. The layer thickness used for measuring has not been given (I), but the samples have probably been diluted to allow measurement in 1.0 cm layers. In this case the measurement may have been influenced by the presence of stray light. Van Assendelft used toluene haemolysis (5) without further purification and measured, without further dilution, in layers of 0.005 cm. The remaining differences are, however, slight, so that the values given in Table 2 and 3, second columns, may be used in the equations given by Benesch et al. (1) without further study. For determining the relative concentrations of Hb, HbO,, and Hi, the equations as given by Benesch et al. (1) could of course be used. Nevertheless when calculating absolute concentrations (mmol * 1-l) these equations must be corrected using the corrected E values given in the second column of Tables 2 and 3.
0.609 0.610 0.613 0.623 0.638 0.658 0.682 0.710 0.746 0.788 0.835 0.882 0.926 0.964
6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8
0.583 0.583 0.586 0.596 0.610 0.629 0.652 0.679 0.714 0.754 0.799 0.844 0.886 0.922
2
0.373 0.374 0.379 0.389 0.405 0.424 0.450 0.48 I 0.520 0.567 0.616 0.665 0.710 0.749
1
EXTINCTION
0.357 0.358 0.363 0.372 0.387 0.406 0.430 0.460 0.497 0.542 0.589 0.636 0.679 0.716
2
COEFFICIENTS
0.355 0.356 0.365 0.380 0.400 0.425 0.455 0.492 0.537 0.590 0.647 0.703 0.755 0.801
I
(t x
IO-‘)
TABLE
3
0.340 0.341 0.349 0.363 0.383 0.407 0.435 0.471 0.514 0.564 0.619 0.672 0.722 0.766
2
OF HAEMIGLOBIN
0.358 0.360 0.370 0.385 0.406 0.433 0.465 0.507 0.558 0.620 0.682 0.745 0.800 0.850
I
I’ The measurements were made in 0.035 M phosphate buffer from pH 6.2 to 7.4 and in 0.1 acid alkaline transition of ferrihaemoglobin is influenced by ionic strength, the ionic strength At pH 7.4 the extinction coefficients were the same in the two buffers. I-Values given by Benesch ef rrl. (1) 2-Values of column 1. corrected for &:,‘[., = I1 .O. 3-Value given by Van Assendelft (5).
I
PH
MOLAR
M
1
pH”
0.412 0.410 0.409 0.406 0.401 0.393 0.380 0.360 0.324 0.306 0.280 0.254 0.230 0.208
OF
0.394 0.392 0.391 0.388 0.384 0.376 0.363 0.344 0.310 0.293 0.268 0.243 0.220 0.199
2
I
) (0.370)
3
Tris buffer from pH 7.4 to 8.8. Since the pK of the was kept constant at 0.1 by the addition of NaCl.
0.342 0.344 0.354 0.368 0.388 0.414 0.445 0.485 0.534 0.593 0.652 0.713 0.765 0.813
2
AS A FUNCTION
48
VAN
ASSENDELFT
AND
ZIJLSTRA
REFERENCES 1. Benesch, R. E., Benesch, R., and Yung, S. (1973) Anal. B&hem. 5.5, 245. 2. Drabkin, D. L., and Austin, J. H. (1935) J. Biol. Chern. 112, 51. 3. Drabkin, D. L. (1961) in Haematin Enzymes (Falk, J. E.. Lemberg. R., and Morton, K., eds.), p. 144, Pergamon Press, New York. 4. International Committee for Standardization in Haematology (1967) Brit. J. Haernnt. 13, Suppl.. 71. 5. Van Assendelft, 0. W. (1970) Spectrophotometry of Haemoglobin Derivatives, Royal Van Gorcum Ltd., Assen, the Netherlands. 6. Meyer-Wilmes, J., and Remmer, H. (1956) Arch. Exp. Pathol. Pharmakol. 229, 441. 7. Remmer, H. (1956) Arch. Exp. Pathol. Pharmakol. 229, 450. 8. Minkowski, A., and Swierczweski, E. (1959) irr Oxygen Supply to the Foetus (Walker, J., and Turnbull, A., eds.). Blackwell, Oxford. 9. Zijlstra, W. G., and Van Kampen, E. J. (1960) Clin. Chim. Acta 5, 719. IO. Wootton, 1. D. P., and Blevin, W. R. (1964) Lancet 2, 434. 11. Van Oudheusden, A. P. M., Van de Heuvel, J. H., Van Stekelenburg, G. J., Siertsema, L. H., and Wadman. S. K. (1964) Ned. T. Geneesk. 108, 265. . 12. Salvati, A. M.. Tentori, L., and Vivaldi, G. (1965) Clin. Chim. Acta 11, 477. 13. Tentori, L., Vivaldi, G., and Salvati, A. M. (1966) Clin. Chim. Acta 14, 276. 14. Morningstar, D. A., Williams, G. Z., and Suutarinen, P. ( 1966) Amer. J. Clin. P&ho/. 46, 603. 15. Stigbrand, T. (1967) Stand. J. Clin. Lab. Invest. 20, 251. 16. Itano, H. A. (1972) in Modern Concepts in Hematology (Izak, G.. and Lewis, S. M., eds.), p. 26, Academic Press, New York/London.
69, 43-48 ( 1975)
BIOCHEMISTRY
Extinction
Coefficients
Spectrophotometric
for
Analysis
Use in Equations
for
the
of Haemoglobin
Mixtures
0. W. VAN ASSENDELFT AND W. G. ZIJLSTRA Laboratory
ofChernica1
Physiology,
Bloernsingel
10, Groningen,
The Netherlands
Received January 38. 1975: accepted June 5, 1975 Values of the extinction coefficients for deoxyhaemoglobin, oxyhaemoglobin. carboxyhaemoglobin, as well as those of haemiglobin between pH 6.2 and 8.8, as published by Benesch et al. (I ), are corrected. based on the internationally accepted value of I I .O for c$&.
Benesch, Benesch, and Yung ( 1) published values for molar extinction coefficients at a wavelength (h) of 540, 560, 576, and 630 nm for deoxyhaemoglobin (Hb), oxyhaemoglobin (HbO,), carboxyhaemoglobin (HbCO), and haemiglobin (ferrihaemoglobin; Hi). All haemoglobin concentrations were calculated in their study by measuring after conversion to haemiglobincyanide and using a millimolar extinction coefficient (E) at A = 540 nm (E.#$.) of 11.5. This value for E.&& had been published by Drabkin (2,3). The International Committee for Standardization in Haematology (ICSH) has, however, published recommendations for the determination of haemoglobin in human blood (4) in which a value of 1 1.0 is given for E#&. As can be seen from Table 1, where results obtained over the past 20 yr by means of various techniques are summarized, this value for E&‘& is indeed based upon abundant experimental evidence. On behalf of those wishing to use the equations given by Benesch et al. (1) for the spectrophotometric analysis of haemoglobin mixtures, the E values from their Tables 1 and 2 have been recalculated on the basis of E&;& = 1 I .O. The results are given in Tables 2 and 3. In the first column the values as published by Benesch et al. are given, in the second column these values corrected for E&$!.!. = 11.0. In the third column the values obtained by Van Assendelft (5) are included in parentheses for comparison. 43 Copyright All rights
Q 1975 by Academic Press. Inc. of reproduction in any form resewed.
Morningstar er al. (14)
Tentori et al. (13)
Wootton and Blevin (10) Van Oudheusden et nl. (11) Salvati et al. (12)
Minkowski and Swierczewski (8) Zijlstra and Van Kampen (9)
Meyer-Wilmes and Remmer (6) Remmer (7)
Author
E&
TABLE
1
Human Hb, toluene haemolysis Human whole blood Human whole blood Human Hb purified on CMC column Human Hb purified on CMC column Human whole blood Human washed cells
Material
0.03 0.07
11.02 10.97
10.99 11.06 10.95
0.05
0.05 0.08 0.03
10.68’
10.90
0.01 0.03 0.02 0.04
10.99 10.94 11.05
OR CARBON
Human Hb, toluene haemolysis
NITROGEN,
0.04 0.04 0.03 0.065 -
ON IRON,
11.0 11.0 11.09 11.19 11.15b
BASED
Horse Hb Horse Hb Human whole blood Human whole blood Foetal whole blood
AUTHORS,
s/(n)1’2
BY DIFFERENT
l&
AS REPORTED
10 6
55
10 8 46
123 35 101 14
12 12 11 4 5
n
CL,a’-dipyridyl 01, a’-dipyridyl TiCI:, ferric perchlorate
Fe; Fe: Fe; Fe;
Fe; X-ray emission spectrography
N analysis
Fe; (Y, a’-dipyridyl Fe; (Y, a’-dipyridyl Fe; a, a’-dipyridyl
o-phenanthroline TiCI, TIC!, complexon o-phenanthroline
Method Fe; Fe; Fe; Fe; Fe;
DETERMINATION’
?i u N t: L
5
B 9 Fz
>
(16)
-
n,“t
error
1 (ntot - 1)
standard
= standard
(15)
(Zi(rli
- 1)
tot
calculated
(.‘i, - k,,)‘),
= 0.01:
II = number
SF + II,
$$
of the mean:
of the mean
error
using
Total
10.88
11.00
the equation
of determinations.
Human Hb purified on CMC or Sephadex column or by dialysis against Na,-EDTA Human Hb A purified by chromatography mean
value
= 10.99:
calculated
using
the equation
C analysis
16
0.04
for E j?&
Fe; sulfosalicylic
55
0.02
acid
11~ = numwhere.r, = mean value of all determinations:\-, = mean value of the ith series of determinations; n ,,)t = total number of determinations: ber of determinations in the ith series; s, = standard deviation of the ith series of determinations; xtOf = standard deviation of all determinations. value was not published. ’ Excluded from calculation of .Y,, and s,,>, because s/(n)” the series differs more than three times the standard error of the mean from the total c Excluded from calculation of S, and stUt because mean value.
n,
&_
Total
l’.~/(h)l’L
Itano
Stigbrand
ii 2 WY
8
f
5
;1 o n
7
46
VAN
ASSENDELFT
MOLAR Deoxy A
1
540 560 570 576 630
1.08 1.34 1.16 1.01 0.115
l-Values ‘-Values A = 540 nm, 3-Values
EXTINCTION
Hb 2
1.03 1.28 1.11 0.97 0.11
AND
TABLE 2 COEFFICIENTS Oxy
3 (1.03) (1.27) (1.10) (0.98) (0.10)
1 1.53 0.906 1.23 1.65 0.015
ZIJLSTRA
(E x 10mJ)
Hb 2
1.46 0.867 1.18 1.58 0.014
given by Benesch et ~1. (1). of column 1 corrected for the millimolar equal to 11.0. given by Van Assendelft (5).
CO Hb 3 (1.43) (0.85) (1.19) (1.53) (0.02)
extinction
1 1.44 1.22 1.51 1.13 0.023
coefficient
2
3
1.38 1.17 I .44 1.08 0.022
(1.43) (1.21) (1.42) (1.12) (0.02)
of HiCN
at
When comparing the values reported by Benesch et al., after correction, with the values reported by Van Assendelft, there remain but slight differences. The values given by Van Assendelft are marginally lower in the case of HbO,; the values given by Benesch et al. are somewhat lower for HbCO. The reason for this is not quite clear. A possible explanation, however, could be found in differences in preparing the samples and possibly in differences in measuring. Benesch et al. not only washed, lysed, and centrifuged to obtain their haemoglobin solutions, but also removed the organic phosphates. The layer thickness used for measuring has not been given (I), but the samples have probably been diluted to allow measurement in 1.0 cm layers. In this case the measurement may have been influenced by the presence of stray light. Van Assendelft used toluene haemolysis (5) without further purification and measured, without further dilution, in layers of 0.005 cm. The remaining differences are, however, slight, so that the values given in Table 2 and 3, second columns, may be used in the equations given by Benesch et al. (1) without further study. For determining the relative concentrations of Hb, HbO,, and Hi, the equations as given by Benesch et al. (1) could of course be used. Nevertheless when calculating absolute concentrations (mmol * 1-l) these equations must be corrected using the corrected E values given in the second column of Tables 2 and 3.
0.609 0.610 0.613 0.623 0.638 0.658 0.682 0.710 0.746 0.788 0.835 0.882 0.926 0.964
6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8
0.583 0.583 0.586 0.596 0.610 0.629 0.652 0.679 0.714 0.754 0.799 0.844 0.886 0.922
2
0.373 0.374 0.379 0.389 0.405 0.424 0.450 0.48 I 0.520 0.567 0.616 0.665 0.710 0.749
1
EXTINCTION
0.357 0.358 0.363 0.372 0.387 0.406 0.430 0.460 0.497 0.542 0.589 0.636 0.679 0.716
2
COEFFICIENTS
0.355 0.356 0.365 0.380 0.400 0.425 0.455 0.492 0.537 0.590 0.647 0.703 0.755 0.801
I
(t x
IO-‘)
TABLE
3
0.340 0.341 0.349 0.363 0.383 0.407 0.435 0.471 0.514 0.564 0.619 0.672 0.722 0.766
2
OF HAEMIGLOBIN
0.358 0.360 0.370 0.385 0.406 0.433 0.465 0.507 0.558 0.620 0.682 0.745 0.800 0.850
I
I’ The measurements were made in 0.035 M phosphate buffer from pH 6.2 to 7.4 and in 0.1 acid alkaline transition of ferrihaemoglobin is influenced by ionic strength, the ionic strength At pH 7.4 the extinction coefficients were the same in the two buffers. I-Values given by Benesch ef rrl. (1) 2-Values of column 1. corrected for &:,‘[., = I1 .O. 3-Value given by Van Assendelft (5).
I
PH
MOLAR
M
1
pH”
0.412 0.410 0.409 0.406 0.401 0.393 0.380 0.360 0.324 0.306 0.280 0.254 0.230 0.208
OF
0.394 0.392 0.391 0.388 0.384 0.376 0.363 0.344 0.310 0.293 0.268 0.243 0.220 0.199
2
I
) (0.370)
3
Tris buffer from pH 7.4 to 8.8. Since the pK of the was kept constant at 0.1 by the addition of NaCl.
0.342 0.344 0.354 0.368 0.388 0.414 0.445 0.485 0.534 0.593 0.652 0.713 0.765 0.813
2
AS A FUNCTION
48
VAN
ASSENDELFT
AND
ZIJLSTRA
REFERENCES 1. Benesch, R. E., Benesch, R., and Yung, S. (1973) Anal. B&hem. 5.5, 245. 2. Drabkin, D. L., and Austin, J. H. (1935) J. Biol. Chern. 112, 51. 3. Drabkin, D. L. (1961) in Haematin Enzymes (Falk, J. E.. Lemberg. R., and Morton, K., eds.), p. 144, Pergamon Press, New York. 4. International Committee for Standardization in Haematology (1967) Brit. J. Haernnt. 13, Suppl.. 71. 5. Van Assendelft, 0. W. (1970) Spectrophotometry of Haemoglobin Derivatives, Royal Van Gorcum Ltd., Assen, the Netherlands. 6. Meyer-Wilmes, J., and Remmer, H. (1956) Arch. Exp. Pathol. Pharmakol. 229, 441. 7. Remmer, H. (1956) Arch. Exp. Pathol. Pharmakol. 229, 450. 8. Minkowski, A., and Swierczweski, E. (1959) irr Oxygen Supply to the Foetus (Walker, J., and Turnbull, A., eds.). Blackwell, Oxford. 9. Zijlstra, W. G., and Van Kampen, E. J. (1960) Clin. Chim. Acta 5, 719. IO. Wootton, 1. D. P., and Blevin, W. R. (1964) Lancet 2, 434. 11. Van Oudheusden, A. P. M., Van de Heuvel, J. H., Van Stekelenburg, G. J., Siertsema, L. H., and Wadman. S. K. (1964) Ned. T. Geneesk. 108, 265. . 12. Salvati, A. M.. Tentori, L., and Vivaldi, G. (1965) Clin. Chim. Acta 11, 477. 13. Tentori, L., Vivaldi, G., and Salvati, A. M. (1966) Clin. Chim. Acta 14, 276. 14. Morningstar, D. A., Williams, G. Z., and Suutarinen, P. ( 1966) Amer. J. Clin. P&ho/. 46, 603. 15. Stigbrand, T. (1967) Stand. J. Clin. Lab. Invest. 20, 251. 16. Itano, H. A. (1972) in Modern Concepts in Hematology (Izak, G.. and Lewis, S. M., eds.), p. 26, Academic Press, New York/London.