SPECTROPHOTOMETRY OF HEMOGLOBIN: A COMPARISON OF DOG AND MAN
WILLEM G. ZULSTRA and ANNEKE B u t m s ~ Department of Physiology, University of Groningen, Bloemsingel 10, 9712 KZ Groningen,
The Netherlands (Tel: 050-632690)
(Received 14 November 1986)
Abstract--l. The absorptivity at 540 nm of hemiglobincyanide (~cN) from dog blood was determined on the basis of iron and found to be within the range formerly obtained for human hemoglobin.
2. Consequently, ~ c N = I1.0, the established value for human hemoglobin, may be used for dog hemoglobin.
3. On this basis the absorption spectra of oxyhemoglobin, de-oxygenated hemoglobin, carboxy- hemoglobin, hemiglobin (methemoglobin) and hemiglobincyanide were determined for dog hemoglobin.
4. No significant differences were found between dog and human hemoglobin, except that dog hemiglobin binds less OH- as reflected in a difference between the absorption spectra of dog and human hemiglobin at the same pH.
INTRODUCTION
Since the absorptivity of human hemiglobincyanide (cyanmethemoglobin; H iCN) at 2 = 540 nm (E~CN) has been established on the basis o f iron by various methods, as well as on the basis of nitrogen and carbon (for a review of the literature see Van Assendelft and Zijlstra, 1975; Van Kampen and Zijlstra, 1983), the absorption spectra of human hemoglobin and its common derivatives have been determined on this foundat ion (Van Assendelft, 1970; Van Kampen and Zijlstra, 1983; Zijlstra et aL, 1983, 1985), and the absorptivities have been used in vari- ous applications (Brown, 1980; Zwart et al., 1981a, 1984). In spectrophotometry of hemoglobin deriva- tives in the blood of other mammals , it was tacitly assumed that the absorptivities as determined for human blood would also be valid for the blood of these animals. However, the finding of small differences between the absorption spectra of adult and fetal human oxyhemoglobin (Zwart et al., 1981b; Huch et al., 1983; Zijlstra et al., 1984), drew attention to the fact that the assumption o f equality between the absorption spectra of the hemoglobins of the common laboratory animals and those o f man was not well-founded and thus might be the cause of considerable analytical error in spectrophotometric mul t icomponent analysis.
As the dog (Canis familiaris) is much used as an experimental animal in studies involving spectro- photometr ic determinations of hemoglobin and its common derivatives, we studied the spectrophoto- metric properties of dog hemoglobin, starting with the
Address for correspondence: Prof. Dr. W. G. Zijlstra, Fysiologisch Laboratorium, Bloemsingel 10, 9712 KZ Groningen, Nederland
determination of E~CN on the basis of iron. Fo r a better comparison between the spectra o f dog and human hemoglobin, we repeated some of our earlier measurements of human blood using exactly the same analytical procedures as those applied to dog blood.
MATERIALS AND METHODS
Blood was obtained from healthy dogs of either sex, which did not receive any drugs, and from healthy human donors. The samples were collected in iron-free Erlenmeijer flasks containing 1 ml heparin solution (ca 5 rag). The erythrocytes were washed three times with 9 g/1 NaCI solu- tion, using iron-free tubes. Then 1 vol. distilled water and 0.4 vol. toluene were added to 1 vol. of erythrocytes. After proper mixing and storing at 4°C for at least 16 hr, the contents of the tube separated into three layers of which the lowest was a clear stroma-free hemoglobin solution. The hemoglobin stock solution thus obtained was centrifuged for 20 rain at 8000g, filtered through an ash-free paper filter and stored at 4°C.
The iron content of the hemoglobin stock solutions was determined with the ~,a'-dipyridyi method (Zijlstra and Van Kampen, 1960). Using a calibrated Ostwald pipet, I ml hemoglobin solution was transferred to each of five iron-free decomposition tubes to which 1 ml HNO 3 65%, 1 ml H202 30% and three drops octylalcohol (octanol-1) were added. The temperature was slowly raised to 220°C on an electric furnace till a clear yellow solution was obtained, which was then evaporated almost to dryness. To remove excess acid, 1.5 ml water was added and evaporated in vacuo at 95°C; this was repeated once. The contents of the decomposition tubes were then transferred to volumetric flasks and made up to 100 ml with water. Four millilitres of this solution was mixed with I ml buffered ~,a'-dipyridyl solution (0.5g ~,a'-dipyridyl, 3.4 g sodium acetate trihydrate and 1.41 ml glacial acetic acid, made up to 50 ml with water) and 1 ml sodium sulfite solution (2.52g Na2SO3.7H20 made up to 100 ml with water). After adding 1.5 ml water the mixture was heated to 95-100°C for 10 rain, while the tubes were covered with glass marbles and the upper ends of the tubes were cooled by an air stream. Absorbance of the pink solution was then measured at 2 = 520 nm on an Optica CF4 spectrophotometer with a lightpath length of 1.000 cm.
251
252 WILLEM G. ZULSrRA and A ~ r d ~ BUURSMA
Appropriate corrections were made for traces of iron in reagents and glassware (blank decomposition) and for the faint yellow background colour of the solution. A cali- bration curve was made using ferric ammonium sulfate, NH4Fe(SO4)2" 12 H20, the iron content of which had been checked by gray±metric analysis, and which was stored in an atmosphere corresponding to the water vapour pressure of the crystals (0.8 kPa). All reagents used were of analytical grade.
For the determination of the absorbance of HiCN at :. = 540 nm, I ml of hemoglobin stock solution was trans- ferred to a volumetric flask with the help of the same Ostwald pipet as used in the iron determination, and made up to 250 ml with a reagent solution containing 200 mg K3Fe(CN)6, 50mg KCN and 1.0g NaHCOa per litre. Absorbance at ~ = 5 4 0 n m (.4 54°) was measured on an Optica CF4 spectrophotometer with a lightpath length of 1.000cm, using the same cuvettes as used in the iron determination. Water was used as a blank, since A 540 of the l:eagent solution is zero. The same HiCN solutions were subsequently used to determine the complete absorption spectra of HiCN.
The millimolar absorptivity (referring to one heine group plus globin moiety) of HiCN at 2 = 540nm was then calculated with the equation:
AU° x V, (1) E HiCN
V2 x cHb(Fe) x l
where V~ is the volume contained by the volumetric flask (250 ml), V 2 is the volume delivered by the Ostwald pipet (I.023ml), cHb(Fe) is the hemoglobin concentration in mmol/1 as determined on the basis of the iron content and l is the lightpath length (1.000 cm).
Deoxygenated (uniiganded) hemoglobin was prepared by tonometry of about 10 ml stock solution with a humidified N2/CO 2 (94.4/5.6%) gas mixture in a revolving glass tono- meter (Rispens et al., 1968). The volume of the tonometer was about 300 ml and the gas flow about 2.5 i/hr. The time required to attain complete deoxygenation was ca 2 hr. Oxygenated (HbO2) and carboxygenated hemoglobin (HbCO) were prepared in a similar way, using O2/CO2 (94.4/5.6%) and CO/N2 (5/95%) gas mixtures, respectively, for tonometry. Hemiglobin (methemoglobin; Hi) was pre- pared by adding five drops of a saturated K3Fe(CN) 6 solution to 10 ml stock solution with a total hemoglobin concentration (Cab.) of 80-100 g/L
Absorbance measurements were made using an Optica CF4 grating spectrophotometer and a Hewiett-Packard HP8450A diode array spectrophotometer. The Optica CF4 was operated with a nominal band width of 0.1 nm and a lightpath length (l) of 0.015 era. This lightpath length was obtained by inserting a 0.085 em plan parallel glass plate
Table 2. Principal peaks and troughs in the absorption spectra
Hemoglobin Dog species Max/min ,l (nm) N E ~
into a 0.100 cm cuvette (Van Kampen and Zijlstra, 1965). A similar cuvette filled with water was used as a blank. In the vicinity of the peaks and troughs in the absorption spectra readings were taken at 0.5 nm intervals. In other spectral regions larger intervals, up to 10nm, were used. The HP8450A was used to determine the complete absorption spectra between 450 and 800 nm with I = 0.015 cm.
All absorbances were reduced to 1= 1.O00cm and Crib. = 1.00 retool/l, i.e. at each wavelength the mill±molar absorptivity was calculated, c~. of each hemoglobin solu- tion was determined by complete conversion to HiCN of the hemoglobin contained in an exactly determined volume, and measuring A~°; e.b. was then calculated on the basis of ~54°.cN = 11.0.
For Hi and HiCN this procedure yields correct values for E x. For Hb, HbO 2 and HbCO the measured values ofE a have to be corrected for any contaminating Hi. This was accom-- plished by the following procedure. The Hi fraction (F~) of each sample was determined with the aid of a cyanide addition method (Dijkhuizen et al., 1977), and the absorp- tivity of derivative X was calculated with the equation:
£~ £~" - - FHi'EHi (2)
l - - FHi
where ¢~ is the millimolar absorptivity of Hb, HbO 2 or HbCO at wavelength 2, ~ ~. the absorptivity of Hi and ~ a the absorptivity as measured for the Hb, HbO 2 or HbCO solution.
RESULTS
~ c N was de termined for the b lood of nine dogs and 11 men. O f each b lood specimen four to six
Table I. Mill±molar absorptivity of HiCN at 2 = 540 nm for dog and man
Values are means _ SEM. Measurements made at room temperature (20-24°C) using an Opt±ca CF4 grating spectrophotometer with a nominal bandwidth of 0,1 nm, e~ in mmolfl per cm; significance of difference between dog and man assessed by Student's t-test, two tailed: *P < 0.05; **P < 0.01.
Hb of dog
Table 3. Isosbestic points in the absorption spectra of Hb, HbO2, HbCO and Hi for blood of dog and man
Hi /HiCN 516.1 8.08 515.6 7.96 597.0 3.05 596.8 3.16
Measurements at room temperature (20-24°C) using an Optica CF4 grating spectrophotometer.
separate determinations were made giving a total of 43 independent measurements for dog blood and 54 independent measurements for human blood. The results are consolidated in Table 1. The wavelengths of the principal peaks and troughs in the absorption spectra of Hb, HbO2, HbCO, Hi and HiCN and the corresponding millimolar absorptivities for the blood of both dog and man are presented in Table 2. The absorptivities have first been calculated for each absorption ~pectrum separately; thereafter mean
and man 253
values and SEM were calculated and the statistical significance of the differences between dog and man were determined using Student's t-test for unpaired samples. The data of Table 3, showing the isosbestic points in the absorption spectra of the various hemo- globin species, were calculated in a different way. First, the mean values of the spectra were calculated, the number of measurements of each hemoglobin species being the same as given in Table 2. Then the crossover points of the mean 2E curves were deter- mined. Therefore no spread of the data is given in Table 3. The absorption spectra of the various hemo- globin species as recorded with the diode-array spec- trophotometer are given in Fig. 1. The corresponding 2( curves of human blood have not been added; the absorption spectra of the various hemoglobin species of man and dog, except those of Hi, are so similar that the minute differences cannot be properly shown in such a presentation. Therefore Table 4 has been added, in which part of the data of Fig. 1 is presented in numerical form next to the corresponding data for human blood. Figure 2 shows the absorption spectra of Hi at pH = 7.15 for dog and man.
DISCUSSION
The two series of 54o Etec~ measurements shown in Table 1 yield mean values of 10.96 and 10.93 for the blood of dog and man, respectively. Although this difference is only 0.3%, it is statistically significant (P < 0.01). The internationally established value of E~CN = 11.0 is based on 14 series of measurements in l0 different laboratories by means of various meth- ods (cf. Table 1 in Van Kampen and Zijlstra, 1983). When the series comprising less than 15 independent determinations are left out, the results spread from 11.05 + 0.02 (SEM; n = 101) based on Fe analysis by means of TiCl3 titration, to 10.90+0.05 (SEM; N = 55) and 10.88 + 0.04 (SEM; N = 16) based on N and C analysis, respectively. The three large series on
E = absorptivity in mmol/1 per cm, measured at room temperature (20-24°C) using an HP8450A diode array spectrophotometer.
the basis of Fe analysis by the ~t,~t'-dipyridyl method taken together yielded Em54°-cN = 10.97--+0.01 (SEM; N = 204). Since there were no arguments for prefer- ring any of the methods used, it was decided to take the weighed mean value of the 14 series of mea- surements (10.99) as the reference value for the international HiCN standard, and thereby as the basis of all hemoglobin spectrophotometry. As our present results arc within the range of values on which the established value for EmeNd° is based, there is no reason to depart from this rule and not to use this value for dog blood as well. Consequently, E~CN = 11.0 has been used in calculating all absorp- tivities presented in Tables 2-4.
For the accurate determination of the peaks and troughs in the absorption spectra of the hemoglobin species, measurement with a very low spectral band width is necessary. Therefore we used for this purpose an Optica CF4 grating spectrophotometer with the nominal band width adjusted to 0.1 nm. The wave- length scale had beforehand been checked with the
help of Hg emission lines. Determination of complete absorption spectra over the visible range is very laborious when a conventional spcctrophotometer like the Optica CF4 is used. Therefore, when such a spectrophotometcr is employed in multicomponcnt analysis only a limited number of wavelengths is used, usually no more than there arc components in the mixture to bc analyzed (Zwart et al., 1981a). A diode array spcctrophotomcter like the HP8450A allows the determination of complete absorption spectra in a few seconds, but at the cost of a considerable loss in spectral resolution. In the HP8450A the diodes arc placed at even wavelengths with an inter-diode distance of 2 nm. The obvious disadvantage is that sharp peaks at odd wavelengths cannot bc faithfully recorded. The important advan- tage, however, is that multicomponent analysis can easily be carried out on the basis of quasi-continuous measurement over the whole visible range (Zwart et al., 1984). For these reasons, wc used both types of spectrophotometer. The equality of the' absorbancc
10.0
-'-" 8.0
60
- ~ ~.0
~ 2.0
I).0
. . . . 50 45( 500 550 600 650 700 7 800
WAVELENGTH (nm)
Fig. 2. Absorption spectra of hemoglobin at pH = 7.15 from dog (dots) and from human bemiglobin (solid line).
Hb of dog and man 255
measurements by the two instruments was chocked with a certified carbon yellow filter made available by the National Bureau of Standards (USA).
Table 2 shows that the spectral properties of most hemoglobin species of dog and man are very much alike; neither in the position of the principal peaks and troughs nor in the absorptivities at these Wave- lengths are appreciable differences found. Even where these differences reach the level of probable statistical significance (P < 0.05), they arc small enough to be of little practical importance. The only exception is in the Hi spectra, where the absorptivities at the two maxima are significantly (P < 0.01) higher in the dog than in man; the difference is 3.0% at g = 500 nm and 3.6% at 2 = 631 nm.
The similarity of the absorption spectra of dog and human hemoglobin is also shown in Table 3, in which are presented the isosbestic points in the absorption spectra of the different hemoglobin species and the corresponding absorptivities. These data may be useful in wavelength selection for various spectre- photometric methods, e.g. when the sum of the concentrations of two hemoglobin species is to be measured.
As shown in Fig. 1 and Table 4, the data obtained by means of the HP8450A are essentially equivalent with those of the Optica CF4. It should be noted that the ~-pcak of HbO~ at 2 = 577nm as shown in Fig. 1 is necessarily too low because the HP8450A actually measures at ,1. = 576 and 578 nm (cf. Table 2). A complete print-out of the absorption data from 2 = 450-800nm with 2 nm increments is available from the authors on request.
The difference between the absorption spectra of Hi of dog and man at the same pH, as shown in Fig. 2, closely resembles the difference between the absorption spectra of human Hi at different pH. Thus it is reasonable to assume that in both cases the difference between the two absorption spectra is caused by a different ratio of Hi and HiOH. Con- sequently, the difference between dog and human Hi must be in the equilibrium constant of the formation of HiOH from Hi, the affinity of dog Hi for O H - being lower than that of human Hi.
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