Mary S. Stern

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

The amino acid sequence of hemoglobin III from the symbiont-harboring clam Lucina pectinata.

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedMm of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem.265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.

Comparison of the hemoglobins of the platyhelminths Gastrothylax crumenifer and Paramphistomum epiclitum (Trematoda: Paramphistomatidae)

1. Gastrothylax crumenifer and Paramphistomum epiclitum parasitize the water buffalo Bubalus bubalis. 2. Gastrothylas hemoglobin consisted of two fractions of ca 30,000 and ca 18,000 by gel filtration. SDS-electrophoresis showed both to be single, ca 15,000 chains. 3. Paramphistomum hemoglobin was ca 16,000 by both gel filtration and SDS-electrophoresis. 4. Reversed-phase chromatography of carboxymethylated trematode and buffalo globins gave single peaks and two peaks, respectively. Although Paramphistomum hemoglobin provided and N-terminal sequence, Gastrothylax hemoglobin did not, suggesting blocked N-terminals. The buffalo sequences were found to be identical to the sequences of the alpha and beta chains of bovine hemoglobin. 5. Although Paramphistomum hemoglobin consists of only one chain, Gastrothylax hemoglobin consists either of one chain which aggregates to a dimer or of two different chains, only one of which aggregates to a dimer.

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated Mm of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem.10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.

Purification and properties of the hemoglobins of the platyhelminth Isoparorchis hypselobagri (trematoda: Isoparorchidae) and its host Wallagu attu (catfish)

1. The hemoglobins of the trematode Isoparorchis hypselobagri and of its host Wallagu attu (catfish) were isolated and purified. 2. SDS-polyacrylamide gel electrophoresis showed both to consist of single, 15-17 kDa chains, having different electrophoretic mobilities. 3. Isoelectric focusing showed the trematode hemoglobin to be homogeneous with a pI of 4.2 and the host hemoglobin to consist of several components. 4. Gel filtration of freshly prepared trematode hemoglobin revealed one peak corresponding to M(r) approximately 17 kDa; gel filtration of a preparation which had been stored for 2-3 months demonstrated the presence of two peaks, whose elution volumes corresponded to M(r) of ca 35 and 17 kDa, respectively. 5. Reversed-phase chromatography of carboxymethylated 35 and 17 kDa peaks on a C8 column, gave a single peak a and two peaks b and c, respectively. 6. Edman degradation of peaks a, b and c obtained provided identical sequences of 27 amino acid residues for peaks a and c and another sequence differing at 10 of the 27 positions, for peak b. Edman degradation of the freshly prepared Isoparorchis hemoglobin provided the first 15 amino acid residues found for peaks a and c. The host hemoglobin gave an N-terminal sequence completely different from the trematode sequences. 7. Since gel filtration of the 35 and 17 kDa peaks showed no sign of an interconversion equilibrium, it appears that the 35 kDa peak and peak a represent a disulfide-bonded dimer of a monomer globin chain which shares the 27 N-terminal residues with chain c, the major monomer globin component of the 17 kDa peak.(ABSTRACT TRUNCATED AT 250 WORDS)

The cDNA Sequences Encoding the Polymeric Globins of Glycera dibranchiata

The nucleated erythrocytes of the marine polychaete Glycera dibranchiata, the bloodworm, contain several globin chains of ca. 17 kDa, separable into two Hb fractions (1-3): a “monomeric” fraction and a “polymeric” fraction consisting of self-associated globin chains. Both fractions are present in individual erythrocytes (4). The amino acid sequences of two “monomeric” Glycera globins have been determined, M-II (5) and M-IV (6), and the crystal structure of M-II (7) has been refined recendy to a 1.5 A resolution (8). In the “monomeric” globins, the distal His (E7) is replaced by a Leu residue. Furthermore, the two fractions exhibit extensive heterogeneity with at least five or six distinct components in the “monomeric” fraction (9-15) and a similar number of components in the “polymeric” fraction (16,17).

Partial Amino Acid Sequence of Hemoglobin II from Lucina pectinata

The Puerto Rican clam Lucina pectinata, a bivalve mollusc, has three cytoplasmic Hbs associated with its gill: HbI, HbII and HbIII. The clam utilizes these three Hbs in symbiosis with intracellular chemoautotrophic bacteria (1). These bacteria reside in the gill of the clam where its requirement for O2 and hydrogen sulfide is provided by the clam via the Hbs (2)