Significance Amyloid folds, while performing functional roles in most domains of life, remain a key factor in the emergence and development of multiple neurodegenerative disorders in humans. The significance of our study is twofold: first, by structurally characterizing highly divergent natural prion amyloids, we uncovered that functional amyloids can evolve in a regime of fold conservation, withstanding extreme sequence diversification. Second, we found that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that key side-chain positions could determine the seeding specificity of an amyloid fold. This work thus sheds light on the fundamental properties of a major category of pathogenic agents.

Fibrinogen-420 is a minor subclass of human fibrinogen that is so named because of its higher molecular weight compared to fibrinogen-340, the predominant form of circulating fibrinogen. Each of the two Aα chains of fibrinogen-340 is replaced in fibrinogen-420 by an Aα isoform termed αE. Such chains contain a globular C-terminal extension, αEC, that is homologous with the C-terminal regions of Bβ and γ chains in the fibrin D domain. The αEC domain lacks a functional fibrin polymerization pocket like those found in the D domain, but it does contain a binding site for β2 integrins. Electron microscopy of fibrinogen-340 molecules showed the major core fibrinogen domains, D–E–D, plus globular portions of the C-terminal αC domains. Fibrinogen-420 molecules had two additional globular domains that were attributable to αEC. Turbidity measurements of thrombin-cleaved fibrinogen-420 revealed a reduced rate of fibrin polymerization and a lower maximum turbidity. Thromboelastographic measurements also showed a reduced rate of fibrin-420 polymerization (amplitude development) compared with fibrin-340. Nevertheless, the final amplitude (MA) and the calculated elastic modulus (G) for fibrin-420 were greater than those for fibrin-340. These results suggested a greater degree of fibrin-420 branching and thinner matrix fibers, and such structures were found in SEM images. In addition, fibrin-420 fibers were irregular and often showed nodular structures protruding from the fiber surface. These nodularities represented αEC domains, and possibly αC domains as well. TEM images of negatively shadowed fibrin-420 networks showed irregular fiber borders, but the fibers possessed the same 22.5-nm periodicity that characterizes all fibrin fibers. From this result, we conclude that fibrin-420 fiber assembly occurs through the same D–E interactions that drive the assembly of all fibrin fibrils, and therefore that the staggered overlapping molecular packing arrangement is the same in both types of fibrin. The αEC domains are arrayed on fiber surfaces, and in this location, they would very likely slow lateral fibril association, causing thinner, more branched fibers to form. However, their location on the fiber surface would facilitate cellular interactions through the integrin receptor binding site.

The scanning transmission electron microscope (STEM) at Brookhaven National Laboratory* (BNL) is nearly unique in its ability not only to image isolated unstained and unshadowed biological molecules but also to obtain quantitative information about them such as their oligomeric state (1) (see Note 1). This makes it ideal, in principle, for looking at protein-DNA complexes and obtaining information about the masses bound. However, there are very stringent requirements on the purity and stability of the samples.

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-β protein (Aβ40ARC) as well as the effect of Aβ40WT on the distribution of the protofibrillar species formed by Aβ40ARC by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Aβ oligomerization and fibrillogenesis in vitro. In addition, Aβ40ARC was observed to affect both the morphology and the size distribution of Aβ protofibrils. Electron microscopy examination of the protofibrils formed by Aβ40ARC revealed several morphologies, including: (1) relatively compact spherical particles roughly 4–5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18–25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Aβ40ARC protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Aβ40WT/Aβ40ARC, suggesting that co-incubation of Aβ40ARC with Aβ40WT leads to kinetic stabilization of Aβ40ARC protofibrils. An increase in the ratio of AβWT/AβMUT(Arctic), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.

There is an ongoing controversy concerning whether crosslinked gamma chains in fibrin are oriented "transversely" between fibril strands or "end-to-end" along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked gamma chains in fibrin under physiological conditions were also aligned end-to-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template. We evaluated the roles of E polymerization sites E(A) and E(B), and D association sites gammaXL, Da, Db, betaC and alphaC in this process. E(A):Da interactions caused fibrinogen: E "DED" complexes to form, and markedly enhanced the gamma chain crosslinking rates of fibrinogen or des alphaC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des alphaC-fibrinogen was still slower. These events showed that although alphaC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the E(A):Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to betaC:betaC contacts resulting from D subdomain rearrangements initiated by the E(B):Db interactions, and also occurred in mixtures of des alphaC-fibrinogen with E-des AB. In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a half-staggered arrangement, an alignment that we attribute to crosslinking of gammaXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand gamma chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated gamma chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned gamma chains.

Trichocyte intermediate filaments (IF) are the principal components of epidermal appendages such as hair and nail. Based on studies by a variety of techniques, it has been inferred that trichocyte IF are structurally similar to other kinds of IF. However, some basic structural attributes have yet to be established: in particular, it has remained unclear whether IF are hollow. We have examined trichocyte IF isolated from rat vibrissae and human hair follicles by electron microscopy. Scanning transmission electron microscopy of freeze-dried specimens yielded mass-per-unit-length values of ∼32 kDa/nm, with the human preparations also containing filaments at half this density, corresponding to two rather than four protofibrils. Radial density profiles calculated from cryo-electron micrographs of vitrified specimens preserved in a near-native state revealed a low-density region of ∼3 nm diameter around the filament axis. A minor species of filament with the same internal structure was surface-decorated with material arranged with a helical pitch length of 9.3 nm. These filaments appear to represent IF coated with associated proteins—perhaps, “high-sulfur” proteins—readied for incorporation into the filament-matrix biocomposite of the mature hair.

Pick's and Alzheimer's diseases are distinct neurodegenerative disorders both characterized in part by the presence of intracellular filamentous tau protein inclusions. The tight bundles of paired helical filaments (PHFs) of tau protein found in Alzheimer's disease (AD) differ from the tau filaments of Pick's disease in their morphology, distribution, and pathological structure as identified by silver impregnation. The filaments of Pick's disease are loosely arranged in pathognomonic spherical inclusions found in ballooned neurons, whereas the tau pathology of AD is classically described as a triad of neuropil threads, neurofibrillary tangles, and dystrophic neurites surrounding and invading plaques. In this study we used the high-resolution technique of scanning transmission electron microscopy to characterize and compare the filaments found in Pick's disease with those found in AD. In addition, we determined the mass/nm length and density of arachidonic acid-induced in vitro-assembled filaments. Three morphologically distinct populations of Pick's filaments were identified but each was indistinguishable from AD-PHFs in mass/nm length and density. Filaments assembled in vitro from single isoforms were similar in mass/nm length, but less dense than AD-PHFs and Pick's disease filaments. Finally, we provide clear structural evidence that a PHF, whether found in disease or assembled in vitro, is composed of two distinct intertwined filaments.

Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, AU55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved.

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to =75% in 1-10 mM Ca(II). Reassembly to HBL structures whose linker contents, STEM images and masses were similar to the native Hb was observed with all the linkers (>/=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

Paired helical filaments (PHF) are abnormal, approximately 20–25-nm wide periodically twisted filaments, which accumulate in Alzheimer's disease (AD) brain and other neurodegenerative disorders, including corticobasal degeneration (CBD). PHF are primarily composed of highly phosphorylated tau protein. However, both phosphorylated and non-phosphorylated forms of tau are able to assemble in vitro into filaments similar in the ultrastructural appearance to PHF. In the present study, filaments were assembled in vitro from unmodified recombinant human tau and the physical mass per unit length of filaments and the mass density were determined using scanning transmission electron microscopy (STEM). Two general types of filaments were observed. One type was composed of 11.4 nm-wide, 10–75 nm long, frequently twisted and PHF-like filaments, with a mass per unit length (44 kDa/nm) approximately one third of that observed in isolated AD filaments. The other were straight filaments, approximately 6.8-nm wide and 0.2–2 μm long, which often formed parallel clusters of two or more filaments. Triple clusters were 19.2-nm wide and had a mass per unit length (70 kDa/nm) approximately two thirds of that seen in isolated AD filaments. Despite different morphology, both twisted and straight filaments had mass densities between 0.48–0.55 kDa/nm3. These values are significantly higher than those reported for PHF found either in AD (0.40 kDa/nm3) or CBD (0.33 kDa/nm3). These results suggest that the packing of tau differs in vivo from that observed in vitro and that specific tau isoform content, elongation of tau molecules by phosphorylation or other factors may be required to reproduce pathological assembly. Therefore mass density determinations appear to be an important criterion in comparing various filaments.

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment—the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-Å resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10–15 Å across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40–45 Å, compared with 25–30 Å in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

Nuclear import of DNA is a central event in genetic transformation of plant cells by Agrobacterium tumefaciens. Agrobacterium elicits tumors on plant hosts by transporting a single-stranded (ss) copy of the bacterial transferred DNA (T-DNA) from its Ti (tumor-inducing) plasmid into the plant cell nucleus. Presumably, the process of T-DNA nuclear import is mediated by two agrobacterium proteins, VirD2 and VirE2, which are thought to directly associate with the transported T-DNA. Both proteins have been shown to contain functional nuclear localizations signals (NLS). Recently, VirE2 alone has been shown to actively transport ssDNA into the plant cell nucleus. To understand the process of DNA nuclear import, it is important to know the structure of the transport intermediate. To this end, complexes of VirE2 and ssDNA were analyzed by scanning transmission electron microscopy (STEM). This analysis suggests that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure. The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import. Detailed mass analysis of VirE2-ssDNA filaments is presented and a structural model is proposed.

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gamma XL"). We investigated the contribution of COOH-terminal regions of fibrinogen Act chains to the gamma XL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D-1, which lack COOH-terminal A alpha chain regions comprising similar to 100, similar to 390, and 413 residues, respectively, The cross-linking rates were I-2 > I-9 > I-9D = D-1, and indicated that the terminal 100 or more ACL chain residues enhance gamma XL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alpha C" region of its A alpha chain (A alpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis, In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the A alpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alpha C" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain, In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal A alpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alpha C domains, leads to increased gamma XL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome." (J. Clin. Invest. 1996. 97:2342-2350.)

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGiant Hexagonal Bilayer HemoglobinsJean N. Lamy, Brian N. Green, André Toulmond, Joseph S. Wall, Roy E. Weber, and Serge N. VinogradovView Author Information Laboratoire des Protéines Complexes, CNRS URA 1334, Université de Tours, 37032 Tours, France, Micromass UK Limited, 3 Tudor Road, Altrincham, Cheshire WA14 5RZ, UK, Equipe d'Ecophysiologie, Station Biologique, UPMC-CNRS-INSU, BP 74, 29682 Roscoff, France, Biology Department, Brookhaven National Laboratory, Upton, New York 11973, Department of Zoophysiology, Institute of Biological Sciences, Aarhus University, 8000 Aarhus C, Denmark, and Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201 Cite this: Chem. Rev. 1996, 96, 8, 3113–3124Publication Date (Web):December 19, 1996Publication History Received31 January 1996Revised2 August 1996Published online19 December 1996Published inissue 1 January 1996https://doi.org/10.1021/cr9600058Copyright © 1996 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views320Altmetric-Citations78LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (428 KB) Get e-AlertscloseSUBJECTS:Chemical structure,Cooperativity,Dissociation,Monomers,Peptides and proteins Get e-Alerts

Fibrinogen ‘Cedar Rapids’ is a heterozygous dysfibrinogenemia characterized by delayed and abnormal fibrin polymerization. The specific molecular defect (γ R275C) is relatively common, but in only one case, fibrinogen Tokyo II, has the ultrastructural basis for defective clot formation been determined. This report reflects similar structural studies on Cedar Rapids fibrinogen and fibrin. Crosslinked fibrinogen molecules and fibrils, were prepared at 1 mg/ml in the presence of factor XIIIa (100 u/ml). When γ chains had become ˜10 to 20% crosslinked to γ dimers, samples were diluted with Hepes buffered saline, pH 7, to a fibrinogen concentration of 5 to 10 μg/ml. Three μl was then injected into 3 μl buffer on a carbon-coated EM grid, the specimen allowed to attach for one minute, fluid-exchanged several times with 150 mM NH4 acetate solution, frozen in liquid nitrogen, freeze-dried, and imaged at the Brookhaven STEM facility using a 40 kv probe focused at 0.25 nm. Fibrin for scanning EM (SEM) was formed directly on carbon-formvar coated gold grids. Clots that had formed overnight were fixed with 2.5% glutaraldehyde in 0.1 M Hepes, pH 7 buffer containing 0.2% tannic acid, washed with buffer, dehydrated, CO2 critical point dried, coated with 7.5 nm platinum, and imaged in a JOEL Field Emission SEM operated at 5 kV.

The dissociation of the similar to 3500-kDa hexagonal bilayer (HBL) hemoglobin (Db) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39](8-) (SiW), [NaSb9W21O86](18-) (SbW) and [BaAs4W40O140](27-) (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (similar to 200 kDa), T+L (similar to 50 kDa), and M (similar to 25 kDa) (T = disulfide-bonded trimer of chains a-c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm . SCN > Gdm . Cl > urea > Gdm . OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed similar to 10-nm particles with masses of 200 kDa, suggesting them to be dodecamers (a + b + c)(3)d(3). OxyHb dissociations in urea and Gdm . Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (less than or equal to 10%) without Group IIA cations. During reassembly, maximal (similar to 60%) at 10 mM cation, D occurs at constant levels (similar to 15%), implying the dodecamer to be an intermediate.

Sedimentation equilibrium measurements and scanning transmission electron microscopy (STEM) mass mapping of the extracellular, hexagonal bilayer hemoglobin (HBL Hb) of the earthwormLumbricus terrestrisprovided masses of 3.41 to 3.66 MDa and 3.56 (±0.13) MDa, respectively. The Hb also contains 57.2 (±6.0) moles of tightly bound Ca per mole of protein. The Hb and its subunits obtained by dissociation, in native, dehemed and reduced carbamidomethylated forms, were subjected to electrospray ionization mass spectroscopy (ESI-MS). Maximum entropy deconvolution identified three groups of peaks, at ∼6 kDa, 24 to 32 kDa and ∼53 kDa corresponding to the monomer subunit M (globin chaind), four linker subunits and the disulfide-bonded trimer T (globin chainsa+b+c). Subunit M consisted of three components,d1(15, 992.4),d2(15, 978.0) andd3(15, 962.1) (±1.0 Da), with relative intensities 1.0:0.5:0.3, respectively. Subunit T consisted of four major components,T1 (52, 922.6),T2 (52, 760.0),T3 (52, 598.5) andT4 (52, 435.4) (±4.0 Da), with relative intensities 0.6:1.0:0.2:0.7, respectively. ESI-MS of carbamidomethylated T, demonstrated that, unlike chainsb(16, 254.4) andc(17, 289.2), chainaexists as a series of four, hexose-connected, glycosylated isoforms,a1 toa4 (19, 389.9, 19, 227.4, 19, 065.3 and 18, 902.9) (±1.0 Da). The mass differences between the deglycosylated chaina(17, 524.0) anda1 toa4 correspond to glycan side-chains (GlcNAc)2(Man)n(n=6 to 9). Four groups of peaks were observed in the 24 to 32 kDa region. LinkersL1a(27, 540.8) andL1b(27, 702.4) (±2.0 Da) are isoforms ofL1(25, 837.5 inN-deglycosylated Hb) with glycan side-chains (GlcNAc)2(Man)n(n=8,9). LinkersL2(32, 104.3 (±5.0) Da) andL3(24, 912.9 (±2.0) Da) occur as single species. LinkersL4atoL4c(24, 019.0, 24, 102.3 and 24, 169.9) (±2.0 Da) with relative intensities 1.0:0.8:0.8, have not been identified previously. From ESI-MS relative intensities,L1:L2:L3:L4=0.6:0.4:1.0:0.5 and globin linker=0.78:0.22. HPLC ofLumbricusHb provided a globin linker=0.73:0.27 (±0.02) and a heme content of 2.52 (±0.14) wt%. A model is proposed for the HBL structure, wherein 12 213.4 kDa dodecamers (144 globin chains, 2561 kDa) decorate a hexagonal framework of 36 linker chains (12L1+ 6L2+ 12L3+ 6L4) to provide a total mass of 3.531 MDa, each dodecamer being in contact with three linker subunits.