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1 Biology Department, Lamar University, PO Box 10037, Beaumont, Texas 77710
2 Department of Biology, Clemson University, 132 Long Hall, Clemson, South Carolina 29634
3 Marine Biomedical Laboratory, Duke Marine Laboratory, Duke University, 135 Duke Marine Lab Road, Beaufort, North Carolina 28516
* To whom correspondence should be addressed. E-mail: christenab{at}hal.lamar.edu
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Abbreviations: RBCs, red blood cells • WVS, water vascular system
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Introduction |
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H. elongata is often found in the lower intertidal zone in protected, low-energy areas of the southeast coast of the United States (Hendler et al., 1995), and the sediments it inhabits are often soft, poorly oxygenated and may contain hydrogen sulfide (Windom and Kendall, 1979; Camargo, 1982; unpubl. data). The distribution of H. elongata is sporadic, but densities in a given area may be as high as 2000/m2 (Valentine, 1991a). The juveniles of H. elongata often settle out onto the arms of the adults (adult disc diameter 5 to 12 mm) (Mortensen, 1920; Valentine, 1991a, b), crawl down the arms of the adult, and grow in the burrow until they reach a size at which they can establish their own burrows. Recently settled juveniles (disc diameter 
0.48 mm) also possess RBCs. Although juveniles smaller than 0.43 mm apparently do not have RBCs (unpubl. data), the stage at which they start producing RBCs is unknown.
Only three of the approximately 2000 species of brittle stars possess hemoglobin: Ophiactis virens (Foettinger, 1880; Cuenot, 1891), Hemipholis elongata (Hajduk and Cosgrove, 1975; Heatwole, 1981; Beardsley et al., 1993), and Ophiactis simplex (Christensen, 1998). Of these three, only H. elongata burrows; the other species inhabit the fouling communities of rock jetties and pilings. As for the functional and biochemical properties of ophiuroid hemoglobins, very little has been reported in the literature (Hajduk and Cosgrove, 1975; Beardsley et al., 1993; Christensen, 1998; Weber and Vinogradov, 2001). Hajduk and Cosgrove (1975) reported that the hemoglobin of H. elongata has a high affinity for oxygen (P90 = 9 mmHg) and is composed of five components separable by acrylamide gel electrophoresis. They also reported two components with molecular weights of 19,000 and 23,000 Da separable by SDS gel electrophoresis. The hemoglobins of the holothurians, the other group of extant echinoderms possessing the iron-containing pigment, have been more thoroughly investigated (Terwilliger and Terwilliger, 1988 [review]; Suzuki, 1989; Mauri et al., 1991; McDonald et al., 1992; Baker and Terwilliger, 1993; Kitto et al., 1998).
The goal of the present study was to characterize the functional, biochemical, and structural properties of the hemoglobins of Hemipholis elongata in more detail. Comparisons were made with the findings of Hajduk and Cosgrove (1975), and the functional and structural properties observed in this study were compared to those of the holothurian hemoglobins.
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Materials and Methods |
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Preparation of hemolysates and determination of total hemoglobin per animal
RBCs were extracted by cutting the animal into small fragments in a small quantity of buffered seawater (50 mM TRIS, pH 8.0 at 20 °C) and then rinsing the fragments until no red color was visible. The body fragments were removed and the cell/buffer mixture was placed on ice. The cells were washed three times in fresh buffer. After a final wash, the supernatant was removed and the cell pellet frozen and thawed to lyse the cells. The thawed pellet was resuspended in about 1 ml of 50 mM TRIS, pH 8 at 20 °C in distilled water and centrifuged at 14,000 x g for 5 min to remove cell remnants and debris. The hemolysate was diluted with appropriate buffer to an absorbance of 0.4 to 0.5 OD units at 540 nm to minimize photometric error (van Assendelft, 1970).
For determination of total hemoglobin, the absorption spectrum of the solution was recorded in a Beckman DU-65 spectrophotometer. The hemoglobin concentration, as heme, was calculated using the Beer-Lambert law and the extinction coefficient for human hemoglobin at 542 nm (van Assendelft, 1970). This concentration was multiplied by the total sample volume to give total hemoglobin (as heme) in millimoles, and then divided by animal wet weight to obtain total hemoglobin per gram of wet weight.
Hemolysates used in the oxygen-binding equilibria and the sulfide sensitivity experiments were prepared as above, but with RBCs taken only from excised arms with the aim of avoiding potential interfering effects due to enzymes released from the gut and gonads. Such material may have increased the formation of methemoglobin in the whole animal hemolysates (see Discussion for methemoglobin effects).
Intracellular heme concentration
RBCs collected from an animal as described above were resuspended in isotonic filtered seawater buffered to pH 8.0 at 20 °C with 50 mM TRIS. A few drops of a suspension of polystyrene microbeads (Polysciences, Inc.) of 5.85 µm ± 0.13 µm diameter were added to the cell suspension. A small drop of this mixture was placed on a glass microscope slide, and a glass coverslip was placed on top of the drop. Excess water was wicked away with a tissue until the coverslip was resting on the microbeads, and the cells were flattened between the coverslip and the slide. This technique serves to set the pathlength for the subsequent absorbance measurements (Colacino and Kraus, 1984). The slide was then placed on the stage of the microspectrophotometer (Mangum et al., 1989). The light transmission spectrum through an area of the slide containing only buffer was collected as a reference. Cells were picked at random on the slide, and the transmission spectrum was recorded through each. The absorbance at 540 nm, 560 nm, and 577 nm was computed from light transmission data. The intracellular hemoglobin concentration was calculated as heme concentration using the Beer-Lambert Law and the extinction coefficients for human hemoglobin at the chosen wavelengths (van Assendelft, 1970). Concentrations calculated at the three wavelengths were averaged for each cell. A total of 48 cells were measured (12 cells on each of four slides prepared from the same cell suspension).
Separation of hemoglobins
The presence of multiple hemoglobins was determined using a Pharmacia fast protein liquid chromatography (FPLC) system. Crude hemolysates were collected as previously described and resuspended in 50 mM TRIS, pH 8.0, at 20 °C. Hemolysates from 7–10 individuals were pooled due to the small quantities of hemoglobin collected from each animal. The pooled samples were equilibrated with 50 mM TRIS, pH 8.0, by dialysis. The hemolysates were then loaded onto a DEAE Q Sepharose Hi-Load 2610 column, 60-ml column volume. The resin had been equilibrated with four volumes of 50 mM, pH 8.0, TRIS buffer. A linear salt gradient (50 mM TRIS to 0.075 M NaCl + 50 mM TRIS) was used for elution. The total gradient volume was 300 ml. Fractions were collected at a rate of 6 min per fraction, and absorbance was read at 280 nm (for protein) and 415 nm (hemoglobin peak).
The fractions showing peak absorbance were electrophoresed on a Pharmacia Phast system using a native gel. Samples were run on a 10% acrylamide gel with pH 8.3 electrophoresis buffer and stained with Coomassie blue. A low-porosity stacking gel (upper fraction) was used to sharpen the banding pattern. Samples contained 5–25 µg of protein. Crude hemolysate and human hemoglobin A were electrophoresed as references.
Determination of molecular weight
The molecular weights of the purified hemoglobin fractions were determined by electrospray ionization mass spectrometry. Samples were prepared by the method described by Stevens et al. (1994). Measurements were made on a Fissons-VG BIO-Q triple quadrupole mass spectrometer equipped with a pneumatically assisted electrospray ionization source operating at atmospheric pressure (supplied by VG Biotech, Altrincham, UK).
Hemoglobin spectra and oxygen-binding equilibria in cellulo
A small portion (
5 mm) of the distal end of an arm was excised from an unanesthetized animal and rinsed in buffer to remove any adhering mud. The arm tip was placed in a large depression slide containing a small amount of 0.45-µm filtered isotonic seawater buffered with 50 mM TRIS of the desired experimental pH (7, 8, or 9). The tube feet were manually stimulated to contract, forcing the RBCs out of the cut end of the radial canal. About 50 µl of the buffer/RBC mixture was transferred to a specially designed gas slide (Colacino and Kraus, 1984). The gas slide was placed on the microscope stage of a diode array microspectrophotometer (Mangum et al., 1989). The slide was maintained at a constant temperature (20° ± 0.5 °C) with water pumped from a refrigerated water bath (Forma Scientific). The internal gas tension of the slide was controlled by a gas-mixing flowmeter (Cameron Instrument Co.). The gases were humidified and brought to experimental temperature before flowing through the sample chamber of the gas slide at 100 ml/min. A total of 60 cells taken from 47 animals were used in these experiments.
To examine the equilibrium oxygen-binding characteristics of the hemoglobin in cellulo, cells were exposed to gases at nine oxygen tensions, ranging from 0 to > 150 mmHg (room air). Fractional saturation values were computed from transmitted light intensities (540 nm, 560 nm, and 580 nm) using a two-wavelength modification of a standard analysis (Rossi-Fanelli and Antonini, 1958). The effect of pH on oxygen affinity was determined from oxygen equilibrium experiments conducted at pH 7.0, 8.0, and 9.0. Temperature effects on oxygen binding were determined from oxygen affinity experiments conducted at 10 °C, pH 8.0, and 20 °C, pH 8.0. The heat of oxygenation (
H) was calculated from the van’t Hoff equation.
Oxygen-binding equilibria in vitro
For measurements on crude hemolysates, about 50 µl of the hemolysate was placed in the working chamber of the gas slide along with a 0.5-cm2 piece of monofilament nylon mesh (105-µm mesh opening) (Small Parts, Inc.). A freshly prepared hemolysate solution was used in each experiment because repeated freezing and thawing increased methemoglobin formation (see Discussion for methemoglobin effects). The nylon mesh served to ensure a stable pathlength for light transmission measurements. Oxygen-binding equilibria were measured as previously described for in cellulo measurements at 20 °C. Measurements on purified FPLC fractions were made using standard tonometric techniques at pH 7 at 20 °C.
Oxygen-binding equilibria of juvenile hemoglobin
Oxygen-binding equilibria for hemoglobins of juvenile H. elongata were measured on both isolated cells and intact animals. For the isolated-cell measurements, cells were collected from individuals of disc diameter 1 mm (n = 5) by excising a small portion of an arm in a small quantity of buffer, 50 mM TRIS, pH 8.0, at 20 °C. The cells were loaded onto the gas slide, and the oxygen binding was measured by the method previously described for the adult cells.
For the whole-animal measurements, an intact juvenile was loaded onto the gas slide. The juvenile was anesthetized by placing it in a small quantity of buffer (< 200 µl) containing a few drops of 7% MgCl2. The individual was then placed centrally in the gas slide chamber in a small drop of buffer/MgCl2. Hemoglobin spectra were taken through a tube foot with RBCs in it. Oxygen-binding experiments were conducted as before. Four individuals were measured in this manner.
Hemoglobin sensitivity to sulfide
The effects of hydrogen sulfide on oxygen-binding equilibria were examined using hemolysates prepared as described earlier (hemoglobin 50 mM TRIS in distilled water, pH 8.0 at 20 °C, adjusted to give an absorbance reading of 0.4–0.5 at 540 nm in an 0.5-cm cuvette). Four milliliters of this solution were placed into each of two glass tonometers equipped with 0.5-cm pathlength cuvettes. The initial oxygenated spectra were recorded from 650 nm to 400 nm on a Beckman DU 65 spectrophotometer. The samples were deoxygenated with 99.999% N2, and a deoxygenated spectrum was taken. Then 100 µl of 10 mM Na2S solution was injected into one tonometer with a syringe; 100 µl of deoxygenated buffer was added to the control tonometer. The spectrum was again recorded. Oxygen was gradually introduced in a stepwise fashion by the injection of room air samples after an equal volume had been removed from the tonometers, and the spectra were measured after a 10-min equilibration. This experiment was repeated three times with freshly prepared hemolysates pooled from three to five individuals.
Stopped-flow measurements of ligand kinetics
Kinetics of the hemoglobin-oxygen reaction were estimated using the stopped-flow technique on the crude cell hemolysates suspended in 50 mM HEPES, pH 7, at 20 °C; the crude hemolysates represent pooled samples. Measurements were made for the O2 "off" reaction (dissociation) and the CO "on" reaction (association). The CO "on" reaction was also examined using flash photolysis. The presence of modulator effects was determined by performing the above reactions in the presence of ATP.
Experiments were performed on a Gibson-Durrum stopped-flow apparatus that consists of a Durrum model 13000 light source and monochromator and Durrum model 110 stopped-flow instrument with pneumatic drive. The dissociation constant, koff, was determined by reacting oxyhemoglobin, from crude hemolysates, with sodium dithionite (Na2S2O4) (about 0.5%). The time course of the reaction was monitored by measuring transmitted light intensities.
Light intensity data were collected by a DASAR data acquisition, storage, and retrieval system with a DW-2 interface to a Tektronix 4052 computer. Initial data analyses were carried out with the ASYST program (Macmillan Software Co.) prior to curve-fitting analysis by a nonlinear least-squares program (Johnson et al., 1981).
The carbon monoxide (CO) association reaction was monitored by the stopped-flow technique and flash photolysis. Flash photolysis was performed with dual fast extinguishing (approximately 30 µs) flash tubes and a Xenon Corp. model B micropulser. The subsequent association of CO and the hemoglobin was then monitored as before.
Amino acid sequence of hemoglobin fractions
Partial amino acid sequences of each purified hemoglobin fraction were determined by the automated Edman method using a Porton Instruments PI 2090 integrated micro-sequencing system. The amino acids were identified by visual inspection of hardcopy plots, using retention times from a standard of PTH amino acids.
Proteins were not digested with proteases prior to sequencing. Each analysis used 150–200 pmol of protein. Samples of fractions 1 and 2 were processed for 40 cycles, at which time it became difficult to distinguish the amino acid peaks from the background noise. Fraction 3 was run for 21 cycles.
The partial sequence for fraction 1 was compared to other protein sequences using the MacVector sequence analysis software (Oxford Molecular Group PLC) and the Entrez database (National Center for Biotechnology Information).
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Intracellular hemoglobin concentration and estimation of hematocrit
The RBC hemoglobin concentration, as heme, was 19.5 ± 5.0 mM (mean ± SD, n = 48 cells). Using a mean cell diameter of 9 µm (Hajduk and Cosgrove, 1975; Heatwole, 1981; unpubl. data) and an intracellular hemoglobin concentration of 19.5 mM, the volume of a single cell is 3.8 x 10-7 mm3 and contains 7.4 x 10-12 mmol hemoglobin. If an animal has a total of 8.5 x 10-5 mmol of hemoglobin, it has approximately 1 x 107 cells. The total cell volume is equal to 4.3 mm3; and for an animal with a total WVS volume of 18.1 mm3 (Beardsley and Colacino, 1994), the fraction of the WVS taken up by cells is 0.24.
Separation of the hemoglobins
The hemoglobin is a heterogeneous mixture. Separation of oxygenated crude cell hemolysates by DEAE Q Sepharose FPLC resulted in three fractions that absorb at 280 and 415 nm (Fig. 1). Native gel electrophoresis of the crude hemolysates yielded five bands, two of the major bands corresponding to FPLC fractions 1 and 2, a third major band of uncertain identity, and two minor bands (Fig. 2). Lack of banding in lane 5 (FPLC fraction 3) is attributed to low concentration applied to the gel.
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Molecular weight of hemoglobin subunits
The technique used to determine molecular weight, electrospray mass spectrometry, breaks polymeric hemoglobins into monomeric subunits and causes the heme to dissociate from the protein. The signals for FPLC fractions 1 and 2 indicated that each was composed of a single protein and a heme peak. The molecular weights of the subunits were as follows: fraction 1—16,080, fraction 2—16,119, and fraction 3—16,143. The hemes all had a molecular weight of 616. The differences in the protein weight indicate differences in amino acid composition for the three subunits.
The mass spectrum generated by the third FPLC fraction indicated the possibility of more than one subunit. However, the low concentration of this sample resulted in high-noise mass spectrometry data, making this conclusion uncertain.
Oxygen-binding equilibria in cellulo and in vitro
The hemoglobin of Hemipholis elongata has a moderate affinity for oxygen (P50 = 11.4 mmHg at pH 8.0, 20 °C) (Table 1). The P50 at pH 8.0 is similar to those reported for holothurian hemoglobins (Table 1). The Hill numbers (n) were greater than 1 for both in cellulo and in vitro measurements, indicating cooperativity and functional hemoglobin composed of at least two subunits (a dimer) (Table 1). Then n values are greater for the in cellulo measurements (n = 2.81) than for the crude hemolysates (n = 1.91). The difference in Hill coefficients may be due to concentration differences (19.5 mM in cellulo vs. 0.1 mM in vitro) (see Discussion for concentration effects).
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Values for P50 measured at the three pHs are significantly different from one another (Student’s t test, P = 0.05) (Table 1). Oxygen affinity increases slightly as pH decreases. This is opposite to the usual pH effect. The slope of the Bohr plot is 0.072. The temperature dependence of the oxygen affinity is small (P50 = 8.9 mmHg at 10 °C vs. 11.4 mmHg at 20 °C). The heat of oxygenation (H) is -4.1 kcal/mol.
Oxygen-binding equilibria of juvenile hemoglobin
The hemoglobin of juveniles (disc diameter 0.48 to 0.8 mm) has a higher affinity (P50 = 2.3 mmHg in cellulo and 4.0 mmHg in tube feet of intact animals, pH 8.0 at 20 °C) for oxygen than the adult hemoglobin (P50 = 11.4 mmHg, disc diameter 5 to 12 mm) (see Table 1). This was true for both isolated RBCs and intact animal measurements. The greater apparent P50 for measurements using intact animals may be explained by oxygen consumption of the animal. The PO2 within the tissues is lower than the external PO2 due to oxygen consumption by the tissues. The difference in PO2 can lead to an overestimation of the P50. Even with the overestimation of the juvenile P50, the value is significantly less than that of the adult, indicating distinct functional differences.
Hemoglobin sensitivity to sulfide
Exposure to sulfide caused no changes in the absorption spectrum, and there was no peak at 620 nm; this peak is characteristic of human sulfhemoglobin in the visible region of the spectrum (van Assendelft, 1970; Carrico et al., 1978). There was also no change in the oxygen affinity of the hemolysates in the presence of sulfide (Fig. 3).
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There was no effect of ATP on the CO "on" reaction. However, the presence of ATP caused a small, but statistically significant (P = 0.05) increase in the dissociation rate constant for both phases of the biphasic oxygen "off" reaction of H. elongata hemoglobin (144.3 ± 2.4 s-1 and 24.2 ± 0.2 s-1).
Amino acid sequence of the protein
Figure 4 shows the partial amino acid sequences for the three FPLC fractions of the hemoglobin. The sequences for fractions 1 and 2 differ by eight amino acids. The partial sequence for fraction 3 was identical to that of fraction 2.
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The computed hematocrit value of H. elongata (0.24) is comparable to the tube foot hematocrits of the holothurian S. briareus (0.24, by direct measurement) (Colacino, 1973). These values are greater than the reported hematocrits of the perivisceral fluids of the holothurians Cucumaria pseudocurata (0.032) (Roberts et al., 1984) and Paracaudina chilensis (0.015) (Baker and Terwilliger, 1993), but less than those for humans (0.4) (Guyton, 1991) and other mammals ( 0.4) (Schmidt-Nielsen, 1990).
Structural properties of the hemoglobin
The hemoglobins of H. elongata are a heterogeneous mixture of three components, as evidenced by FPLC. Because the samples were pooled from several individuals, it is not known whether hemoglobin heterogeneity is a normal characteristic of H. elongata blood or an artifact of sample mixing. However, the identical results (e.g., same ratio of fractions) obtained on two separate occasions suggest that all three fractions are present within an individual. Hajduk and Cosgrove (1975) reported only two components separable by gel filtration, one apparently composed of monomers and the other of dimers. The difference in number of fractions may be attributable to difference in separation techniques. Gel filtration separates on the basis of size and shape; DEAE Q Sepharose FPLC (ion exchange) separates molecules on the basis of charge.
Both the present study and Hajduk and Cosgrove (1975) obtained five bands by acrylamide gel electrophoresis. Two of the major bands reported here correspond to FPLC fractions 1 and 2. Because this sample was a crude hemolysate, the two minor bands may have been due to proteins other than hemoglobin, found within or associated with the RBCs. Due to the presence of heme at high relative concentration, two of the major bands from the crude hemolysate were visible on the gel prior to staining with Coomassie blue. The two minor bands were not visible. If the minor bands do represent hemoglobins, the low concentration, as evidenced by the faintness of the bands on the gel, could explain why these fractions were not isolated by FPLC.
The molecular masses of the various fractions (approximately 16,000 Da) are comparable to human ß chain (15,860 Da) (Dickerson and Geis, 1980) and holothurian hemoglobin monomers (17,000–18,000 Da) (Terwilliger and Terwilliger, 1988). These values are smaller than those reported by Hajduk and Cosgrove (1975) (19,000 and 23,000 Da). Differences between the previously reported weights and those of the present study may be attributed to differences in techniques. Mangum (1992) remarked that many of the early studies on holothurian hemoglobin reported similarly large molecular weights that were later found to be smaller (
17,000 Da). She attributed these differences to refinement of molecular techniques.
The cooperative binding of oxygen, both in cellulo and in vitro (Hill number 1), indicates that the functional hemoglobin exists as a polymer. Many of the holothurian hemoglobins are known to exist as dimers (Terwilliger and Terwilliger, 1988) and possibly tetramers (Baker and Terwilliger, 1993). The purified fractions, 1 and 2, of H. elongata hemoglobin have only one globin chain type but exhibit cooperativity in oxygen binding, suggesting that both are able to form cooperative homodimers. The Hill numbers typically observed at low concentrations, somewhat less than 2, are consistent with the formation of cooperative homodimers. Larger assemblies may form under some circumstances, as evidenced by Hill numbers greater than 3 that were observed under some conditions (e.g., in cellulo).
The normally existing homopolymers of several invertebrate hemoglobins exhibit cooperativity (Scapharca inaequivalvis [Chiancone et al., 1981; Royer et al., 1985]; holothurians [Terwilliger and Read, 1972; Bonaventura et al., 1976]), although homodimers and homotetramers of human (and other vertebrate) hemoglobins do not. In fact, the mechanism for cooperativity in invertebrates is thought to be different from that utilized by vertebrates (Riggs, 1998). Cooperativity is thought to be due to interactions between the E and F helices of the hemoglobin subunits, first described in the arcid clam Scapharca inaequivalvis (Royer et al., 1985, 1990). This same association has been described for the innkeeper worm, Urechis caupo (Kolatkar et al., 1994) and a sea cucumber, Caudina arenicola (Mitchell et al., 1995). However, Kitto et al. (1998) believe that the cooperativity mechanism in C. arenicola differs from that in S. inaequivalvis as the residues involved at the crucial contact points are different for the two species. Further structural studies on H. elongata hemoglobin are needed to investigate the nature of the interactions of its subunits.
Comparison of the amino acid sequence of fraction 1 from H. elongata with sequences reported for the globins of the holothurians Caudina arenicola (Mauri et al., 1991; McDonald et al., 1992) and Paracaudina chilensis (Suzuki, 1989) reveals little homology. The lack of homology between the ophiuroid and holothurian globins has contributed to the inability to identify the brittle star globin gene by using holothurian primers (Kitto, pers. comm.). Furthermore, no successful primers for the hemoglobin gene have been generated based on the 39 amino acid sequence of the ophiuroid (Kitto, pers. comm.).
Oxygen-binding characteristics of the hemoglobin
The hemoglobin of H. elongata has a moderate affinity for oxygen, both in cellulo and in vitro. It is not certain at this time whether all of the FPLC fractions exist in the same cell, in separate cell populations, or even within the same animal. However, a large number of cells from many individuals were examined microspectrophotometrically and little variation was seen within the measured P50 values observed within each treatment. This suggests that the different hemoglobins represented by the purified FPLC fractions are present within a single cell. The ratio of this mixture is unknown.
The oxygen affinity results of the present study differ greatly from those reported by Hajduk and Cosgrove (1975) (P90 = 9 mmHg). On the basis of the Hill numbers from the present study, a hemoglobin with a P90 of 9 mmHg (pH 7.2) should have a P50 of about 3 mmHg. The in cellulo and in vitro analysis in the present study demonstrated a P50 between 8 and 9 mmHg at pH 7.0, 20 °C. This difference in oxygen affinities may be attributable to the formation of methemoglobin. The oxygen affinity of mammalian hemoglobin increases with increasing percentages of methemoglobin in the sample (Darling and Roughton, 1942)
Several of the holothurian hemoglobins are prone to oxidization and denaturation at pH 7.0 (Terwilliger and Read, 1972; Bonaventura et al., 1976; Steinmeier and Parkhurst, 1979). When the crude hemolysates of H. elongata were repeatedly frozen and thawed, the P50 decreased. Tests for the presence of methemoglobin showed an increase in the amount present in the sample. Freshly prepared H. elongata hemolysates were typically 5%–7% methemoglobin as determined by the ferrocyanide method (van Assendelft, 1970). In one experiment, the methemoglobin fraction was 13% initially and rose to 45% by the end. The P50 calculated for this run was 3.6 mmHg. This corresponds to the P50 value estimated from the data of Hajduk and Cosgrove (1975).
The formation of methemoglobin may explain the low P50 of the purified fractions. Although it is not unusual for different hemoglobins within an individual to have different binding affinities, the apparent P50 of the mixture usually lies between those of the separate fractions, not above them. During oxygen-binding experiments on the purified fractions, methemoglobin rose from 10% to 16% of the total in fraction 1 and from 32% to 42% in fraction 2. While the large proportion of methemoglobin, particularly in fraction 2, makes comparisons of P50 difficult, the importance of this experiment is the demonstration of the cooperative binding of homopolymers.
Another explanation for the difference in oxygen affinities may be the presence of intracellular modulators that would have been removed during the purification of the fractions. However, many invertebrate hemoglobins, including the holothurian hemoglobins, are insensitive to organic phosphates (Terwilliger and Terwilliger, 1988; Scholnick and Mangum, 1991; Baker and Terwilliger, 1993). The rate constant for the oxygen "off" reaction of H. elongata crude hemolysates exhibited a small, but significant, increase in the presence of ATP.
The differences in apparent cooperativity between the in cellulo and in vitro measurements may be due to concentration effects. Dilute preparations may be less aggregated than concentrated ones, with the consequence that the Hill coefficient increases due to subunit interactions as the concentration of hemoglobin increases. This may account for the fact that the hemoglobins of capitellid worms were reported to have greater cooperativity in cellulo than in vitro (Mangum et al., 1992).
Other than those reported here, no data on ligand-binding kinetics are available for the brittle star hemoglobins. The smaller of the two dissociation rate constants for H. elongata hemoglobin is similar to those of human (40 s-1, Antonini and Brunori, 1971) and holothurian hemoglobin (S. briareus, 4.8 s-1 [Colacino, 1973]; Thyonella gemmata, 3.6 to 8 s-1 [Steinmeier and Parkhurst, 1979]). The larger of the two dissociation rate constants (134.6 s-1) is among the largest reported. The CO association rate constant of H. elongata is between those of holothurian hemoglobin (S. briareus, 103 M-1s-1 [Colacino, 1973]; T. gemmata, 103 to 104 M-1s-1 [Steinmeier and Parkhurst, 1979]) and human hemoglobins (30 x 105 M-1s-1 [Antonini and Brunori, 1971]).
Most hemoglobins exhibit a decrease in oxygen affinity as the pH decreases (Weber, 1980). It is generally accepted that most holothurian hemoglobins are insensitive to pH (Terwilliger and Terwilliger, 1988). A weak pH dependence (normal direction) on the oxygen affinity of hemoglobin has only been reported for two holothurians, Paracauding chilensis (Baker and Terwilliger, 1993) and Molpadia arenicola (Bonaventura et al., 1976). The hemoglobin of H. elongata, however, shows a slight increase in its oxygen affinity, both in cellulo and in vitro, with a decrease in pH (Table 1). The magnitude of the Bohr shift of H. elongata hemoglobin, however, is very small and may not have any adaptive significance under physiological conditions.
There is not a large temperature dependence, as evidenced by the small change in affinity with a change in temperature. The heat of oxygenation, H = -4.1 kcal/mol, is smaller than that reported for other hemoglobins, -6 kcal/mol to -8 kcal/mol (Antonini and Brunori, 1971; Colacino, 1973). The possession of a hemoglobin with a small temperature dependence may be adaptive to an intertidal organism, because it would make it relatively insensitive to the large temperature changes that can occur in the intertidal environment (Cochran and Burnett, 1996; unpubl. data).
The high affinity of the hemoglobin of juvenile brittle stars is clearly different from that of the adults. This functional difference could be adaptive in their normal habitat. The planktonic larvae often settle out onto the extended arms of the adults. The juveniles then crawl down the arm into the burrow of the adult, where they move around within the burrow (pers. obs). Small juveniles (disc diameter < 1 mm) do not extend their arms into the water column as the adults do; therefore, they must obtain oxygen from the burrow environment or from the adult. The measured PO2 of the burrow water is very low (undetectable with FOXY system [Ocean Optics, Inc.]; unpubl. data), but some oxygen must escape into the environment from the adult, as evidenced by the fact that the sediments lining the burrow are oxidized. The possession of high-affinity hemoglobin by the juveniles would aid them in the acquisition of oxygen from this low-oxygen environment. The switch from high-affinity hemoglobin in the fetus/juvenile to lower affinity hemoglobin in the adult has been documented in many animals (Barcroft, 1935; Riggs, 1951). The time in development in H. elongata when the switch from high-affinity to low-affinity hemoglobin occurs is not known. Also unknown is whether the hemoglobin present in the juvenile RBCs is one of the hemoglobins found in the adults or an entirely different one. Due to the small size of the juveniles and the small numbers collected, insufficient hemoglobin has been isolated to make comparisons with adult hemoglobin by gel electrophoresis.
The hemoglobins of H. elongata have a higher affinity for oxygen than those of another ophiuroid species, Ophiactis simplex (Christensen, 1998) (see Table 1). The differences in the oxygen-binding properties could be related to differences in their lifestyles. H. elongata burrows in anoxic mud, does not ventilate its burrow, and lacks genital bursae, structures known to serve as sites of gas exchange in ophiuroids. O. simplex is epibenthic and commonly occurs in fouling communities associated with rock jetties and wharf pilings. The oxygen levels in these environments may not be as limiting as in the mud. The function of hemoglobin in O. simplex is not known, but is under investigation.
Effects of sulfide on oxygen binding
Vertebrate and many invertebrate hemoglobins bind with sulfide to form sulfhemoglobin. In human hemoglobin A, the binding of sulfide takes place at the heme, but not to the iron, and is effectively irreversible (Berzofsky et al., 1971; Carrico et al., 1978). This also decreases oxygen affinity so much as to render the hemoglobin functionless under normal conditions (Carrico et al., 1978). Many invertebrate hemoglobins show reversible binding with sulfide (Arp and Childress, 1983; Childress et al., 1984; Doeller et al., 1988; Somero et al., 1989). In many cases the binding of sulfide is vital to the organism because it harbors endosymbionts that require sulfide as an energy source (Felbeck, 1983; Hand and Somero, 1983; Powell and Somero, 1986). No such endosymbionts have been observed in H. elongata (unpubl. data.)
No abnormal spectral changes were observed during the oxygen equilibrium experiments on H. elongata hemoglobin exposed to sulfide. This indicates that either the hemoglobin is insensitive to sulfide or there are detoxifying enzymes present. However, due to the nature of the hemolysate solution, any enzymes present would be greatly diluted, thus decreasing their efficiency. The lack of abnormal spectra points toward a hemoglobin that is insensitive to sulfide. The hemoglobins of the phoronid Phoronis architecta (Vandergon and Colacino, 1991) and the polychaete Abarenicola affinis (Wells and Pankhurst, 1980) also appear to be insensitive to sulfide. If sulfide does bind to the hemoglobin of H. elongata, it does so in a way that does not alter oxygen affinity or the visible absorption spectra. For organisms that live in sulfide-rich environments but do not rely on sulfide-requiring symbionts, it would be highly adaptive to possess hemoglobin whose function is not disrupted by exposure to sulfide.
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1.7) of the hemoglobin would allow it to extract oxygen from the overlying water column and deliver it to buried body parts over a wide range of external and internal PO2 values. The relative insensitivity of the hemoglobins to changes in temperature and pH preserve hemoglobin function when conditions change, as they frequently do in an intertidal environment. The insensitivity to hydrogen sulfide ensures that the hemoglobins continue to function below the sediment surface where the animal is situated and sulfide levels may be high.
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). Hemoglobin is crude hemolysate suspended in 50 mM Tris, pH 8.0, at 20 °C.