Sedimentation Velocity Analytical Ultracentrifugation Research Articles

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors.

A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Quantifying Trace Amounts of Aggregates in Biopharmaceuticals Using Analytical Ultracentrifugation Sedimentation Velocity: Bayesian Analyses and F Statistics.

Analytical ultracentrifugation-sedimentation velocity (AUC-SV) is often used to quantify high molar mass species (HMMS) present in biopharmaceuticals. Although these species are often present in trace quantities, they have received significant attention due to their potential immunogenicity. Commonly, AUC-SV data is analyzed as a diffusion-corrected, sedimentation coefficient distribution, or c(s), using SEDFIT to numerically solve Lamm-type equations. SEDFIT also utilizes maximum entropy or Tikhonov-Phillips regularization to further allow the user to determine relevant sample information, including the number of species present, their sedimentation coefficients, and their relative abundance. However, this methodology has several, often unstated, limitations, which may impact the final analysis of protein therapeutics. These include regularization-specific effects, artificial "ripple peaks," and spurious shifts in the sedimentation coefficients. In this investigation, we experimentally verified that an explicit Bayesian approach, as implemented in SEDFIT, can largely correct for these effects. Clear guidelines on how to implement this technique and interpret the resulting data, especially for samples containing micro-heterogeneity (e.g., differential glycosylation), are also provided. In addition, we demonstrated how the Bayesian approach can be combined with F statistics to draw more accurate conclusions and rigorously exclude artifactual peaks. Numerous examples with an antibody and an antibody-drug conjugate were used to illustrate the strengths and drawbacks of each technique.

Mechanistic Implications of the Unique Structural Features of the Anti‐Sigma Domain of the Pseudomonas Sigma Regulator, PupR

Gram‐negative bacteria tightly regulate intracellular iron, an essential nutrient. To ensure this strict control, some outer membrane TonB‐dependent transporters (TBDTs) responsible for iron import stimulate their own transcription in response to extracellular binding of ferric siderophore. This process is mediated by an inner membrane sigma regulator protein that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we employ the Pseudomonas putida ferric‐pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X‐ray crystal structure of the cytoplasmic anti‐sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small‐angle X‐ray scattering, and sedimentation velocity analytical ultracentrifugation (SV‐AUC) all indicate that, in contrast to other ASDs, the PupR‐ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by SV‐AUC and thermal denaturation circular dichroism spectroscopy. Additionally, we have demonstrated that the helical content of the PupR‐ASD increases in the presence of trifluoroethanol, and are investigating the interaction of the PupR‐ASD with its cognate sigma factor, PupI. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcriptional activation.Support or Funding InformationFunded by NSF ND EPSCoR DDA #FAR0025216 to JJ; NIH NINDS 1R03 NS090939 and NSF MCB‐1413525 to SS; NIH NIGMS 1R15 GM113227, NIH NCRR 2P20 RR015566, and NIH NIGMS P30 GM103332 to CC.

Gravitational Sweep Sedimentation Velocity

Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by approximately 100-fold through the use of a quasi-continuous increase of rotor speed during the experiment. Such ‘gravitational sweep’ sedimentation approaches has previously been shown to be very effective in the study of nanoparticles with large size ranges. However, previously diffusion processes were not accounted for, therefore posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In the present work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extends the macromolecular size-range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitudes of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, for example, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar sized macromolecular species, in the study of broadly distributed nanoparticles.

Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by ∼100-fold with a quasi-continuous increase of rotor speed during the experiment. Such a gravitational-sweep sedimentation approach has previously been shown to be very effective in the study of nanoparticles with large size ranges. In the past, diffusion processes were not accounted for, thereby posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In this work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extend the macromolecular size range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitude of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar-sized macromolecular species, or in the study of broadly distributed nanoparticles.

Sedimentation coefficient distributions of large particles.

The spatial and temporal evolution of concentration boundaries in sedimentation velocity analytical ultracentrifugation reports on the size distribution of particles with high hydrodynamic resolution. For large particles such as large protein complexes, fibrils, viral particles, or nanoparticles, sedimentation conditions usually allow migration from diffusion to be neglected relative to sedimentation. In this case, the shape of the sedimentation boundaries of polydisperse mixtures relates directly to the underlying size-distributions. Integral and derivative methods for calculating sedimentation coefficient distributions g*(s) of large particles from experimental boundary profiles have been developed previously, and are recapitulated here in a common theoretical framework. This leads to a previously unrecognized relationship between g*(s) and the time-derivative of concentration profiles. Of closed analytical form, it is analogous to the well-known Bridgman relationship for the radial derivative. It provides a quantitative description of the effect of substituting the time-derivative by scan differences with finite time intervals, which appears as a skewed box average of the true distribution. This helps to theoretically clarify the differences between results from time-derivative method and the approach of directly fitting the integral definition of g*(s) to the entirety of experimental boundary data.

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6.

The 20S core particle of the eukaryotic proteasome is composed of two α- and two β-rings, each of which is a hetero-heptamer composed of seven homologous but distinct subunits. Although formation of the eukaryotic proteasome is a highly ordered process assisted by assembly chaperones, α7, an α-ring component, has the unique property of self-assembling into a homo-tetradecamer. We used biophysical methods to characterize the oligomeric states of this proteasome subunit and its interaction with α6, which makes direct contacts with α7 in the proteasome α-ring. We determined a crystal structure of the α7 tetradecamer, which has a double-ring structure. Sedimentation velocity analytical ultracentrifugation and mass spectrometric analysis under non-denaturing conditions revealed that α7 exclusively exists as homo-tetradecamer in solution and that its double-ring structure is disassembled upon the addition of α6, resulting in a 1:7 hetero-octameric α6–α7 complex. Our findings suggest that proteasome formation involves the disassembly of non-native oligomers, which are assembly intermediates.

Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR.

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this strict control, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small-angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation all indicate that, in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.

Effects of Linker Length and Transient Secondary Structure Elements in the Intrinsically Disordered Notch RAM Region on Notch Signaling

Formation of the bivalent interaction between the Notch intracellular domain (NICD) and the transcription factor CBF-1/RBP-j, Su(H), Lag-1 (CSL) is a key event in Notch signaling because it switches Notch-responsive genes from a repressed state to an activated state. Interaction of the intrinsically disordered RBP-j-associated molecule (RAM) region of NICD with CSL is thought to both disrupt binding of corepressor proteins to CSL and anchor NICD to CSL, promoting interaction of the ankyrin domain of NICD with CSL through an effective concentration mechanism. To quantify the role of disorder in the RAM linker region on the effective concentration enhancement of Notch transcriptional activation, we measured the effects of linker length variation on activation. The resulting activation profile has general features of a worm-like chain model for effective concentration. However, deviations from the model for short sequence deletions suggest that RAM contains sequence-specific structural elements that may be important for activation. Structural characterization of the RAM linker with sedimentation velocity analytical ultracentrifugation and NMR spectroscopy reveals that the linker is compact and contains three transient helices and two extended and dynamic regions. To test if these secondary structure elements are important for activation, we made sequence substitutions to change the secondary structure propensities of these elements and measured transcriptional activation of the resulting variants. Substitutions to two of these nonrandom elements (helix 2, extended region 1) have effects on activation, but these effects do not depend on the nature of the substituting residues. Thus, the primary sequences of these elements, but not their secondary structures, are influencing signaling.

Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22

Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1–58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140–162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a “nutcracker” with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.

Characterization of Human Polyamine Oxidase

Polyamine Oxidase (PAO) is a flavoprotein that is necessary for the catabolism of polyamines. PAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine, respectively. Normal levels of polyamines are needed for cell growth, yet their actual function is not well understood. The crystal structures of maize and yeast PAO (Fms1) are available but no mammalian PAO structure has been determined to date. The identity between human PAO (hPAO), Fms1, and maize PAO is only 20%. Sequence homology and mutational analysis showed that a conserved histidine residue in mammalian PAOs that corresponds to position 67 of Fms1 helps to properly position the amine substrate for oxidation. Determining the crystal structure of hPAO will help identify the other residues that play a role in substrate binding and catalysis.hPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. hPAO was expressed and purified using affinity and ion exchange columns. The his‐tag was removed and the protein is being assayed for activity. Analytical ultracentrifugation sedimentation velocity experiments are also underway to determine the oligomerization state of the protein in solution in addition to crystallization trials to determine the structure of hPAO.

Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market.

Elucidating Complicated Assembling Systems in Biology Using Size-and-Shape Analysis of Sedimentation Velocity Data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has seen a resurgence in popularity as a technique for characterizing macromolecules and complexes in solution. SV-AUC is a particularly powerful tool for studying protein conformation, complex stoichiometry, and interacting systems in general. Deconvoluting velocity data to determine a sedimentation coefficient distribution c(s) allows for the study of either individual proteins or multicomponent mixtures. The standard c(s) approach estimates molar masses of the sedimenting species based on determination of the frictional ratio (f/f0) from boundary shapes. The frictional ratio in this case is a weight-averaged parameter, which can lead to distortion of mass estimates and loss of information when attempting to analyze mixtures of macromolecules with different shapes. A two-dimensional extension of the c(s) analysis approach provides size-and-shape distributions that describe the data in terms of a sedimentation coefficient and frictional ratio grid. This allows for better resolution of species with very distinct shapes that may co-sediment and provides better molar mass determinations for multicomponent mixtures. An example case is illustrated using globular and nonglobular proteins of different masses with nearly identical sedimentation coefficients that could only be resolved using the size-and-shape distribution. Other applications of this analytical approach to complex biological systems are presented, focusing on proteins involved in the innate immune response to cytosolic microbial DNA.

A Reason for Long Tales

Transcription Activator-Like Effectors (TALEs) are bacterial virulence factors containing a domain of repeats that recognize specific DNA sequences and reprogram transcription activation of invaded plant cells. TALE genes encode 5 to 30 repeats with average pairwise repeat identities greater than 91%. Most variability arises from only two positions termed Repeat Variable Diresidues (RVDs), which confer DNA binding specificity. Crystal structures of free and DNA-bound TALEs (Deng et al. Science 2012) show a large conformational change upon DNA binding. Thus, DNA binding is likely coupled to the free energy of tertiary structural change between the TALE repeats. We are interested in quantifying this relationship and relating it to folding cooperativity using nearest-neighbor (“Ising”) models.To investigate the length dependence of folding, we created a set of consensus TALE constructs of varying length. Solubilizing N- and C-terminal caps are needed to favor monomeric protein in solution as detected by sedimentation velocity analytical ultracentrifugation. Capped consensus TALE repeat constructs have alpha-helical secondary structure as measured by farUV CD. Urea-induced unfolding transitions of TALE repeat arrays were measured and show cooperative unfolding transitions as well as increases in stability with length. These data are well-fitted by an Ising model, which separates the contributions of intrinsic and interfacial free energies. TALE repeats have an unfavorable intrinsic folding free energy of 5.3 kcal/mol and a favorable interfacial free energy of −6.8 kcal/mol. Using these values, the length dependence of TALE stability can be modeled, and shows TALEs under 5 repeats to be unfolded. Using the Ising parameters, we find that partially folded states with a single repeat unfolded are energetically accessible. Population of these partially folded states may be important for DNA binding.

Native Purification and Analysis of Long RNAs.

The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation-renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2'-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.

Characterization of Capsular Polysaccharides and Their Glycoconjugates by Hydrodynamic Methods.

Hydrodynamic methods are relevant for the characterization of carbohydrates such as capsular bacterial polysaccharides or glycoconjugates in solution. This chapter focuses on the following hydrodynamic methods: sedimentation velocity analytical ultracentrifugation (SV AUC), dynamic light scattering (DLS), sedimentation equilibrium analytical ultracentrifugation (SE AUC), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and capillary viscometry-intrinsic viscosity measurement. The chapter highlights the general principle of these five methods, describes experimental details, and specifies advances in the last years.

Assembly of the RIG-I 2CARD Signaling Complex

The innate immune system of mammals utilizes pattern-recognition receptors to differentiate between pathogen associated molecular patterns and host molecules. The retinoic acid inducible-gene-I (RIG-I) like receptors (RLRs) bind viral dsRNAs, triggering an innate immune response. RLRs contain two N-terminal caspase activation and recruitment domains (2CARD), a DExD/H-box helicase-like domain, and a C-terminal regulatory domain. In the absence of RNA, the 2CARD region is sequestered by the helicase domains in an autoinhibited state. Upon binding to the 5′-ends of activating viral dsRNAs a conformational change exposes 2CARD. The CARD domains then interact with MAVS, a mitochondrial outer membrane protein, leading to downstream signaling and interferon expression. Signaling is modulated by K63-linked polyubiquitin chains (polyUb). It has been reported that polyUb binding to 2CARD induces formation of 2CARD tetramer. However, the nature of the assembly process is not well understood and we are investigating the thermodynamic linkage between polyUb binding and 2CARD oligomerization using sedimentation velocity analytical ultracentrifugation. 2CARD alone exists as a monomer; however, in the presence of polyUb several high molecular weight complexes are formed. The assembly process is dependent on salt, polyUb concentration, and polyUb chain length.

Accounting for Photophysical Processes and Specific Signal Intensity Change in Fluorescence-Detected Sedimentation Velocity Analytical Ultracentrifugation

Fluorescence detected sedimentation velocity (FDS-SV) analytical ultracentrifugation has emerged as a powerful technique for the study of macromolecular interactions, particularly high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. In order to fit the FDS data structure, in the quantitative analysis it is essential to adjust the conventional sedimentation models for detailed description of the sedimentation boundaries. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for sub-diffraction microscopy. At the same time, we find conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the data quality.

Characterization of Amynthas Gracilis Hemoglobin (HbAg) and its Subunits by AUC and MALDI-TOF-MS

The giant extracellular hemoglobin (HbAg) of the annelid Amynthas gracilis has a molecular mass (MM) of 3400kDa. In the current work, the characterization of MM values of HbAg and its subunits is presented. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbAg subunits are very close, but not identical to those of Glossoscolex paulistus (HbGp) and Rhinodrilus alatus (HbRa) hemoglobins. Analytical ultracentrifugation (AUC) sedimentation velocity experiments were performed to obtain M for HbAg in oxy- form. value of 59.3 ± 0.2 S was obtained for native HbAg. From the ratio between sedimentation and diffusion coefficients values for M of approximately 3400 ± 100 kDa for oxy-HbAg was obtained. MALDI-TOF-MS data gave MM for HbAg subunits. Monomer d is found to exist in, at least, four isoforms with MM 16,244±3Da, 16,459±5Da, 16,667±5Da and 16,855±3Da, as noticed for HbGp, and not observed for HbRa. Furthermore, the trimer subunit presents two isoforms ((abc)1 and (abc)2) with MM 51,415±20Da and 51,610±14Da, respectively. This might indicate that the monomers a, b and c do have isoforms, as found for HbGp and not for HbRa. The monomeric chains a, obtained from the trimer abc reduction, present three isoforms with MM 17,015Da, 17,061Da and 17,138Da, differing from HbGp that presents four isoforms. A less intense species is observed at 67,717, and is due to the tetramer abcd contribution. Finally, AUC and MALDI-TOF-MS data are very close as compared to that obtained for HbGp and HbRa. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Finantial support: FAPESP and CNPq Brazilian agencies.