A flexible instrument control and image acquisition system for a scanning electron microscope.

We have developed an instrument control and image acquisition system for use with scanning electron microscopes. By making the system flexible over a wide range of operating voltages, scan generation and image acquisition modes can be easily accommodated to a wide range of instruments. We show the implementation of this system for use with a custom-built low-voltage scanning electron microscope. We then explore the simple modifications that are required for control of two instruments intended for use as free electron lasers.

First tests of a dipole lens for a scanning electron microscope.

We have previously shown that a dipole lens has superior properties that are particularly suited for use in a low voltage scanning electron microscope (SEM) (Tsai & Crewe, 1996). The aberrations are lower than for any other type of lens and lead to a prediction of high resolution. We describe the construction details of a microscope based on this principle and present some early results.

Molecular modeling of the interaction of diagnostic radiopharmaceuticals with receptor proteins: m2 antagonist binding to the muscarinic m2 subtype receptor.

Models of the m2 muscarinic receptor have been built and acetylcholine and an antagonist of the quinuclidinyl benzilate family docked to the putative active site. We have incorporated aspects of homology, site-directed mutagenesis studies and structure-activity studies of specific lead compounds in the construction of our receptor models with a primary focus on the structure of the binding sites. We have observed a deep pocket binding of 5-BrQNT, suggesting a plausible explanation for the observation that agonists and antagonists do not bind competitively. The results of these computational studies are interpreted within the context of the observed in vitro results. Our goal is to assist in the development of subtype receptor selective radiopharmaceuticals for use in PET and SPECT.

Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.

Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.

Adventitious variability? The amino acid sequences of nonvertebrate globins.

1. The more than 140 amino acid sequences of non-vertebrate hemoglobins (Hbs) and myoglobins (Mbs) that are known at present, can be divided into several distinct groups: (1) single-chain globins, containing one heme-binding domain; (2) truncated, single-chain, one-domain globins; (3) chimeric, one-domain globins; (4) chimeric, two-domain globins; and (5) chimeric multi-domain globins. 2. The crystal structures of eight nonvertebrate Hbs and Mbs are known, all of them monomeric, one-domain globin chains. Although these molecules represent plants, prokaryotes and several metazoan groups, and although the inter-subunit interactions in the dimeric and tetrameric molecules differ from the ones observed in vertebrate Hbs, the secondary structures of all seven one-domain globins retain the characteristic vertebrate "myoglobin fold". No crystal structures of globins representing the other four groups have been determined. 3. Furthermore, a number of the one-, two- and multi-domain globin chains participate in a broad variety of quaternary structures, ranging from homo- and heterodimers to highly complex, multisubunit aggregates with M(r) > 3000 kDa (S. N. Vinogradov, Comp. Biochem. Physiol. 82B, 1-15, 1985). 4. (1) The single-chain, single-domain globins are comparable in size to the vertebrate globins and exhibit the widest distribution. (A) Intracellular Hbs include: (i) the monomeric and polymeric Hbs of the polychaete Glycera; (ii) the tetrameric Hb of the echiuran Urechis; (iii) the dimeric Hbs of echinoderms such as Paracaudina and Caudina; and (iv) the dimeric and tetrameric Hbs of molluscs, the bivalves Scapharca, Anadara, Barbatia and Calyptogena. (B) Extracellular Hbs include: (i) the multiple monomeric and dimeric Hbs of the larva of the insect Chironomus; (ii) the Hbs of nematodes such as Trichostrongylus and Caenorhabditis; (iii) the globin chains forming tetramers and dodecamers and comprising approximately 2/3 of the giant (approximately 3600 kDa), hexagonal bilayer (HBL) Hbs of annelids, e.g. the oligochaete Lumbricus and the polychaete Tylorrhynchus and of the vestimentiferan Lamellibrachia; and (iv) the globin chains comprising the ca 400 kDa Hbs of Lamellibrachia and the pogonophoran Oligobrachia. (C) Cytoplasmic Hbs include: (i) the Mbs of molluscs, the gastropods Aplysia, Bursatella, Cerithedea, Nassa and Dolabella and the chiton Liolophura; (ii) the three Hb of the symbiont-harboring bivalve Lucina; (iii) the dimeric Hb of the bacterium Vitreoscilla; and (iv) plant Hbs, including the Hbs of symbiont-containing legumes (Lgbs), the Hbs of symbiont-containing non-leguminous plants and the Hbs in the roots of symbiont-free plants.(ABSTRACT TRUNCATED AT 400 WORDS)

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

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clam Lucina 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-Johnson et 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 A 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 two Lucina 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 two Lucina 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 the Lucina globins and the cytoplasmic hemoglobin of Busycon canaliculatum.

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)

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)

Calculation of subunit stoichiometry of large, multisubunit proteins from amino acid compositions.

The subunit stoichiometry of a large, multisubunit protein can be determined from the molar amino acid compositions (i amino acids) of the protein and its subunits. The number of copies of the subunits (1, 2, ... j) is calculated by solving all possible combinations of simultaneous equations in j unknowns (i!/j!(i - j)!). Calculations carried out using the published amino acid compositions determined by analysis and the compositions calculated from the sequences for two proteins of known stoichiometry provided the following results: Escherichia coli aspartate transcarbamoylase (R6C6, Mr = 307.5 kDa), R = 5.6 to 6.6 and C = 5.8 to 6.3, and spinach ribulose-bisphosphate carboxylase (L8S8, Mr = 535 kDa), L = 7.3 to 9.1 and S = 5.6 to 10.6. Calculations were also carried out with the amino acid compositions of two much larger proteins, the E. coli pyruvate dehydrogenase complex, Mr = 5280 kDa, subunits E1 (99.5 kDa), E2 (66 kDa), and E3 (50.6 kDa), and the extracellular hemoglobin of Lumbricus terrestris, Mr = 3760 kDa, subunits M (17 kDa), D1 (31 kDa), D2 (37 kDa), and T (51 kDa); the results for PDHase were E1 = 20 to 24, E2 = 18 to 31, E3 = 21 to 33 and those for Lumbricus hemoglobin were M = 34 to 46, D1 = 13 to 19, D2 = 13 to 18, and T = 34 to 36. Although the sample standard deviations of the mean values are generally high, the proposed method works surprisingly well for the two smaller proteins and provides physically reasonable results for the two larger proteins.

Novel S-S loops in the giant hemoglobin of Tylorrhynchus heterochaetus.

The extracellular hemoglobin of the polychaete Tylorrhynchus heterochaetus is a "giant" multisubunit protein consisting of two types of subunits: a "monomeric" chain (chain I) and a disulfide bonded "trimer" of chains IIA, IIB, and IIC. We have reported the complete amino acid sequences of all four chains (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). The sites of disulfide bonds in the trimer have now been determined. In the trimer, there are two interchain disulfide bonds between chains IIA and IIC, and IIB and IIC, respectively. In addition, each of the four chains, I, IIA, IIB, IIC, has an intrachain disulfide bond. Thus, according to our "192-chain" model (Suzuki, T., and Gotoh, T. (1986) J. Mol. Biol. 190, 119-123), there are 288 disulfide bonds in Tylorrhynchus hemoglobin. Digital image processing of scanning transmission electron micrographs of negatively stained Tylorrhynchus hemoglobin indicated dimensions of 28 x 18 nm.

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

A fraction obtained by gel filtration at neutral pH of the extracellular Hb of Lumbricus terrestris dissociated either at pH 9.8 or at pH 4.0, consisting of the three subunits D1 (31 kDa), D2 (37 kDa), and T (50 kDa), was found to produce two peaks when subjected to gel filtration on Superose 6 at pH 7. The first peak, which was eluted at a slightly greater volume than the native Hb, consisted of reassociated hexagonal bilayer structures when examined by scanning transmission electron microscopy. The dimensions of the two reassociated hexagonal bilayer structures were a vertex-to-vertex diameter of 25 nm and a height of 16 nm. The difference in size between the hexagonal bilayer structures and the native Hb is the contribution of subunit M, which consists of a single heme-containing chain I (16.75 kDa). Although the reassociated hexagonal bilayer structures have overall dimensions smaller than the 30 nm x 20 nm dimensions of the native Hb, the diameters of the central cavities are not substantially altered. Subtraction of the three-dimensional reconstructions of the reassociated hexagonal bilayer structures from those of the native Hb showed that subunit M was primarily localized at the periphery of Lumbricus Hb. The formation of hexagonal bilayer structures in the complete absence of subunit M provides additional support for the "bracelet" model of the quaternary structure of Lumbricus Hb proposed recently by us in which subunits D1 and D2 were assumed to act as linkers for complexes of subunits M and T or to form a "bracelet" decorated with 12 complexes of subunits M and T.

Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.

The complete dissociation of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and III (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains II, III, and IV) and that peak II consisted of subunit M (16 kDa, chain I). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak Ib (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak Ib contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and III. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumbricus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were approximately equal to 20% smaller than those of the native hemoglobin.

The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure.

Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or "bracelet," decorated with 12 complexes of M and T subunits.

The molecular size of Myxicola infundibulum chlorocruorin and its subunits.

The molecular shape and size of the extracellular chlorocruorin of Myxicola infundibulum was determined using scanning transmission electron microscopy and its dissociation in the presence of sodium dodecyl sulfate (SDS) was investigated using polyacrylamide gel electrophoresis. The shape of the chlorocruorin is that of a two-tiered hexagon with a vertex-to-vertex diameter of 29.0-29.5 nm and a height of 19.0-19.7 nm: it appears to be smaller by 5-10% relative to several annelid extracellular hemoglobins examined by scanning transmission electron microscopy. The quaternary structure of the chlorocruorin appears to be sensitive to Ca(II) concentration; dissociation fragments of the whole molecule were observed, consisting of octamers an dimers of one-twelfth subunits. The unreduced chlorocruorin dissociated into two subunits with estimated molecular masses of 23 000 (1) and 60 000 (2); the reduced chlorocruorin dissociated into subunits with estimated molecular masses of 13 000 (I), 14 000 (II) and 30 000 (III). SDS-polyacrylamide gel electrophoresis of reduced subunits 1 and 2 showed that subunit 1 corresponded to subunit III and that subunit 2 dissociated to subunits I and II. Densitometry of the polyacrylamide gels indicates that 85-90% of the Myxicola chlorocruorin consists of disulfide-bonded tetramers of polypeptide chains of about 15 000. Such a pattern of subunit aggregation has not been observed previously in annelid extracellular hemoglobins and chlorocruorins.

The reassociation of Lumbricus terrestris hemoglobin dissociated at alkaline pH.

The reassociation of the extracellular hemoglobin of Lumbricus terrestris (Mr approximately 3.9 X 10(6) ) at neutral pH, subsequent to its dissociation at pH above 8.0, was examined using gel filtration, ultracentrifugation, and scanning transmission electron microscopy. Gel filtration on Sephacryl S-200 at pH 6.8 of the hemoglobin exposed to pH above 8 showed the presence of four peaks: Ia, consisting of whole molecules, undissociated and reassociated, and smaller heme-containing fragments Ib (Mr approximately 3.0 X 10(5) ), II (Mr approximately 6.5 X 10(4) ), and III (Mr approximately 1.8 X 10(4) ). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that although the pattern of Ia was identical with that of the native hemoglobin, consisting of six polypeptide chains (I-VI), Ib appeared to have less of chains V and VI; II consisted of polypeptide chains II-VI, and III was identified as chain I. Lumbricus hemoglobin exposed to pH over the range 8.4 to 10.2 was subjected to gel filtration on Sepharose CL-6B and resolved into undissociated and dissociated fractions. The combined dissociated fractions, when brought back to pH 6.8 and subjected to gel filtration on Sepharose CL-6B and Sephacryl S-200, demonstrated that the extent of reassociation into whole molecules (IaR) varied from about 50% at pH 8 to 30% at pH 10.2. IaR possessed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern identical with that of the native hemoglobin. Ia, IaR, and Ib dissociated when exposed to alkaline pH; upon return to neutral pH, both Ia and IaR reassociated partially to whole molecules but Ib did not. These results suggest that reassociation comprises two pathways: one leading to the formation of IaR and a "dead-end" pathway, along which reassociation stops at the level of Ib. Digital image processing of scanning transmission electron micrographs of negatively stained native hemoglobin and IaR showed that the two molecules were very similar.

Inexact three-dimensional reconstruction of a biological macromolecule from a restricted number of projections.

A computer-generated three-dimensional model of Lumbricus terrestris hemoglobin has been obtained by reconstruction from four STEM low-dose computer-averaged micrographs. X-ray projections of a wooden model constructed from the calculated two-dimensional sections through the molecule were generally consistent with the original STEM projections of the hemoglobin and suggest the presence of large gaps between the central regions of adjacent 1/12th subunits of opposite tiers.

Subunits of the extracellular hemoglobin of Arenicola marina.

The molecular weight of the extracellular hemoglobin of Arenicola marina determined by equilibrium sedimentation is 3.74 +/- 0.12 . 10(6). Its iron content is 0.244 +/- 0.005 wt.% corresponding to a minimum molcular weight of 22 900 +/- 500. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into three subunits: 14 000 (subunit 1), 31 000 (subunit 2) and 49 000 (subunit 3); in the presence of 2-mercaptoethanol four subunits were observed, 14 000 (subunit I), 16 000 (subunit II), 31 000 (subunit III), and 35 000 (subunit IV). Two-dimensional electrophoresis showed that subunit 1 produced subunit I, and subunits 2 and 3 produced subunits I, II and variable amounts of subunits III and IV. Gel filtration of reduced and alkylated A. marina hemoglobin in 6 M guanidinium hydrochloride suggests that the molecular weight of subunits I and II is 17 500 +/- 1000. A. marina hemoglobin appears to consist of at least 5--7 polypeptide chains. It is proposed that some of the polypeptide chains can associate to form dimers (subunits 2, III, IV) or trimers (subunit 3).

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.