X-ray fluorescence spectrometric determination of the metal content of the extracellular hemoglobin of Tubifex tubifex

Biochimica et Biophysica Acta, 707 (1982) 291-293

291

Elsevier Biomedical Press

BBA Report BBA 30028

X-RAY F L U O R E S C E N C E S P E C T R O M E T R I C D E T E R M I N A T I O N OF T H E METAL C O N T E N T O F T H E EXTRACELLULAR H E M O G L O B I N OF TUBIFEX TUBIFEX M.J. R O K O S Z a and S.N. V I N O G R A D O V b

Analytical Sciences Department, Ford Scientific Research Laboratories, Dearborn, M I 48121 and h Department of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Received May 18th, !982)

Key words: Hemoglobin," Metal content," X-ray fluorescence spectrometry; (Tubifex)

X-ray fluorescence spectrometric analysis of Tubifex hemoglobin, using the energy-dispersive and wavelength-dispersive spectrometers, demonstrated that in addition to Fe, Ca and small amounts of Zn, Cu and Pb were also present. Assuming that Tubi[ex hemoglobin contains 160 atoms of Fe per molecular weight of 3.9- 106, the molar ratios of Fe:Ca: Zn:Cu:Pb were calculated to be 160: 70:2:8:2. The iron content of the hemoglobin was also determined spectrophotometrically using the formation of Fe(II)-l,10-phenanthroline and Fe(II)-4-(2-pyridylazo)-resorcinol complexes: it was found to be 0.240-+0.010% by weight and was unaffected by prior dialysis against 10 mM EDTA solution at neutral pH. Thus, it is unlikely that Tubi[ex hemoglobin contains any non-heine iron.

The properties of the extracellular hemoglobin of the fresh water oligochaete Tubifex tubifex are similar to those of other annelid extracellular hemoglobins [ 1-5]. Although its iron content of 0.22 wt% [1] is in agreement with the known iron contents of other annelid extracellular hemoglobins [6,7], its heme content was found to be about half that of the iron [1]. Such a discrepancy between the iron and heme contents has not been found for any of the oligochaete or polychaete extracellular hemoglobins which have been investigated since 1959, including Lumbricus hemoglobin [6,7]. In the present investigation we report the results of the determination of the iron content of Tubifex hemoglobin by two different spectrophotometric methods as well as by X-ray fluorescence spectrometry. The latter technique is nondestructive and permits the determination of elements other than iron [8]. Our results showed that Ca and small amounts of Cu, Zn and Pb were also present in Tubifex hemoglobin. Live T. tubifex were harvested from the mud 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

banks of the Rouge River. The hemoglobin was extracted by repeated freezing and thawing of the worms in 0.1 M Tris-HC1 buffer, pH 7.2, containing 0.1 m g / m l phenylmethylsulfonyl fluoride. The crude extract was centrifuged at 5000 X g for I h, filtered, made 100 mM in MgC12 and allowed to stand overnight at 5-7°C. After centrifugation at 500 X g for 2 h, the supernatant was centrifuged at 50000 X g. The pelleted hemoglobin was dissolved in the Tris-HCl buffer, passed over a 2 X 20 cm Sepharose B column and pelleted again by highspeed centrifugation. The hemoglobin was dialyzed exhaustively against distilled, deionized water or against 10 mM EDTA, pH 7, followed by dialysis against water. Lyophilized samples of protein solutions were dried to constant weight by heating at 100°C under vacuum, weighed and digested using Cameron's procedure [9]. The concentration of the Fe(II)-l,10-phenanthroline complex was determined from the absorbance at 550 nm using a molar absorptivity of 11000 [9]. The concentration

292

of iron was also determined from the concentration of the Fe(II)-4-(2-pyridoxylazo)-resorcinol complex using an absorptivity at 510 nm of 56000 [10]. All spectrophotometric measurements were performed using a Cary model 15 spectrophotometer. 1,10-Phenanthroline and 4-(2-pyridylazo)-resorcinol were obtained from Eastman Kodak. The X-ray fluorescence analysis of the lyophilized hemoglobin was carried out using a Kevex energy-dispersive spectrometer and a Siemens wavelength-dispersive spectrometer. The energydispersive spectrometer system was composed of a Kevex 0910A sample and secondary target changer, with a Siemens 800 series X-ray generator and a Tracor-Northern analyzer operated by a Digital PDP 11,/05 computer with a dual floppy disc mass storage. The 11/05 was capable of running either the Digital RT-11 operating system when communicating with the wavelength-dispersive spectrometer and central site computers or the Tracor Flex operating system when acquiring sample data. In the latter mode it utilized the Tracor Super ML (XML) program [11]. The spectrometer was operated at 40 kV and 25 mA using a Cr target tube and a Zr secondary target and an 80 mm 2 Si(Li) detector with 180 eV F W H M for MnK radiation; the dead time was approx. 20%. Acquisition times of 1000 s for samples, 5000 s for blanks and 100 s for single-element standards_were used to obtain adequate statistics. The wavelength-dispersive spectrometer system consisted of a Siemens SRS-1 spectrometer with a Canberra computer interface and actuators and a Digital PDP 11,/54 computer. The spectrometer was purged with helium to reduce X-ray optical-path absorption and operated

at 56 kV and 40 mA, using the direct radiation from a Cr target tube to excite the sample spectrum. Data acquisition was automatic, as was the computation of peak and background counting times. These were determined by the software to optimize the counting statistics for each element in each sample. The peak-to-background counting ratio was estimated via a short (5 s) initial counting time and was used to set a total count time (TtotaI) which was then apportioned between peak (Tpeak) and background (Tb) counting times according to the equations: Tpeak = Ttota I >(

(peak count/background count)1/2 l + (peak count/background

T~, = Tlota I

c o u n t ) I/2

Tpeak

which give optimal statistics for a fixed counting time; the peak count refers to a counting time at the mathematically determined centroid position above and below the peak in 20 (energy) previously determined by the analyst. Data from both the energy-dispersive and wavelength-dispersive instruments were merged and transmitted to a central site DEC-20 computer in a form compatible with the N R L X R R program input requirements using hardware and software developed at the Ford Laboratories [12]. Commercially available thin-film, single-element standards (MicroMatter Co., East Sound, WA) were employed. X-ray liquid cells, about 7ml in capacity (No. 3515, Spex Industries, Inc., Metuchen, NJ), in whose closed end a hole about 1 cm in diameter was made, were used for the protein samples. A piece of Kapton film was stretched over the open

TABLE I METAL CONTENT OF T U B I F E X HEMOGLOBIN X-ray fluorescencedata are given as mol relative to Fe; averages of both wavelength-dispersive spectrometer and energy-dispersive spectrometer data. except Ca, which was determined only by wavelength-dispersivespectrometer. 1,10-Ph, 1,10-phenanthroline; PAR, 4-(2-pyridylazo)-resorcinol. Dialyzed

H 20 EDTA, H 20

Weight% Fe

X-ray fluorescence

1,10-Ph

PAR

Fe

Ca

Cu

Zn

Pb

0.243 ÷ 0.010 0.243 -+0.015

0.245± 0.010 0.236--+0.015

160 160

70 65

2 1.6

8 8.4

2 I

293

end of the cell and held in place by a slip-on ring: the protein solution was pipetted into the inverted cell, frozen, taking care that the liquid level inside the cell was horizontal, and lyophilized. The results of the two sets of determinations are shown in Table I. The spectrophotometrically determined iron content of Tubifex hemoglobin is in satisfactory agreement with the earlier result [1] and is in the range of the iron contents of other invertebrate hemoglobins [6,7]. Furthermore, it can be seen that the iron content of Tubifex hemoglobin was essentially unaffected by dialysis against 10 mM E D T A at neutral pH. Since heme iron should not be affected by this treatment our result suggests that non-heme iron is not present in the molecule. The X-ray fluorescence analysis of Tubifex hemoglobin demonstrated that, besides iron, it contains Ca and small amounts of Zn, Cu and Pb. The results are presented as mol relative to 160 mol Fe. Recent studies of the properties of Tubifex hemoglobin, of its dissociation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [5] and of its dissociation at alkaline pH by gel filtration [13] strongly suggest that its size and dimensions are very similar to those of Lumbricus terrestris hemoglobin [14,15]. Hence we assumed that the iron content of Tubifex hemoglobin (0.24%) corresponds to approx. 160 Fe atoms per molecular weight of 3.8 • 10 6. Ca, Zn and Cu have also been found by X-ray fluorescence spectrometry of Lumbricus and Arenicola hemoglobins (M. Rokosz and S. Vinogradov, unpublished results). The presence of Ca in Lumbricus hemoglobin has already been reported [16]: it was estimated that there was one calcium ion per 3.2-3.8 heine groups, i.e., a total number of 42-50 calcium ions, assuming 160 heme groups. This number is comparable to the number of Ca atoms

observed in the present study. Pb is present in amounts equivalent to about 1-2 atoms or less per molecule of Tubifex hemoglobin. Since this element was not found in the other two hemoglobins, its presence in the Tubifex molecule is probably due to the Pb present in the Rouge River. This research was supported in part by US Public Health Service Grant H L 25952.

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