Connectivity in the cold: the comparative population genetics of vent-endemic fauna in the Scotia Sea, Southern Ocean.

We report the first comparative population genetics study for vent fauna in the Southern Ocean using cytochrome C oxidase I and microsatellite markers. Three species are examined: the kiwaid squat lobster, Kiwa tyleri, the peltospirid gastropod, Gigantopelta chessoia, and a lepetodrilid limpet, Lepetodrilus sp., collected from vent fields 440 km apart on the East Scotia Ridge (ESR) and from the Kemp Caldera on the South Sandwich Island Arc, ~95 km eastwards. We report no differentiation for all species across the ESR, consistent with panmixia or recent range expansions. A lack of differentiation is notable for Kiwa tyleri, which exhibits extremely abbreviated lecithotrophic larval development, suggestive of a very limited dispersal range. Larval lifespans may, however, be extended by low temperature-induced metabolic rate reduction in the Southern Ocean, muting the impact of dispersal strategy on patterns of population structure. COI diversity patterns suggest all species experienced demographic bottlenecks or selective sweeps in the past million years and possibly at different times. ESR and Kemp limpets are divergent, although with evidence of very recent ESR-Kemp immigration. Their divergence, possibility indicative of incipient speciation, along with the absence of the other two species at Kemp, may be the consequence of differing dispersal capabilities across a ~1000 m depth range and/or different selective regimes between the two areas. Estimates of historic and recent limpet gene flow between the ESR and Kemp are consistent with predominantly easterly currents and potentially therefore, cross-axis currents on the ESR, with biogeographic implications for the region.

Verbal memory declines more in female patients with Parkinson's disease: the importance of gender-corrected normative data.

BACKGROUND: Data on gender-specific profiles of cognitive functions in patients with Parkinson's disease (PD) are rare and inconsistent, and possible disease-confounding factors have been insufficiently considered. METHOD: The LANDSCAPE study on cognition in PD enrolled 656 PD patients (267 without cognitive impairment, 66% male; 292 with mild cognitive impairment, 69% male; 97 with PD dementia, 69% male). Raw values and age-, education-, and gender-corrected Z scores of a neuropsychological test battery (CERAD-Plus) were compared between genders. Motor symptoms, disease duration, l-dopa equivalent daily dose, depression - and additionally age and education for the raw value analysis - were taken as covariates. RESULTS: Raw-score analysis replicated results of previous studies in that female PD patients were superior in verbal memory (word list learning, p = 0.02; recall, p = 0.03), while men outperformed women in visuoconstruction (p = 0.002) and figural memory (p = 0.005). In contrast, gender-corrected Z scores showed that men were superior in verbal memory (word list learning, p = 0.02; recall, p = 0.02; recognition, p = 0.04), while no difference was found for visuospatial tests. This picture could be observed both in the overall analysis of PD patients as well as in a differentiated group analysis. CONCLUSIONS: Normative data corrected for gender and other sociodemographic variables are relevant, since they may elucidate a markedly different cognitive profile compared to raw scores. Our study also suggests that verbal memory decline is stronger in women than in men with PD. Future studies are needed to replicate these findings, examine the progression of gender-specific cognitive decline in PD and define different underlying mechanisms of this dysfunction.

The biogeography of the yeti crabs (Kiwaidae) with notes on the phylogeny of the Chirostyloidea (Decapoda: Anomura).

The phylogeny of the superfamily Chirostyloidea (Decapoda: Anomura) has been poorly understood owing to limited taxon sampling and discordance between different genes. We present a nine-gene dataset across 15 chirostyloids, including all known yeti crabs (Kiwaidae), to improve the resolution of phylogenetic affinities within and between the different families, and to date key divergences using fossil calibrations. This study supports the monophyly of Chirostyloidea and, within this, a basal split between Eumunididae and a Kiwaidae-Chirostylidae clade. All three families originated in the Mid-Cretaceous, but extant kiwaids and most chirostylids radiated from the Eocene onwards. Within Kiwaidae, the basal split between the seep-endemic Kiwa puravida and a vent clade comprising Kiwa hirsuta and Kiwa spp. found on the East Scotia and Southwest Indian ridges is compatible with a hypothesized seep-to-vent evolutionary trajectory. A divergence date estimate of 13.4-25.9 Ma between the Pacific and non-Pacific lineages is consistent with Kiwaidae spreading into the Atlantic sector of the Southern Ocean via the newly opened Drake Passage. The recent radiation of Kiwaidae adds to the list of chemosynthetic fauna that appear to have diversified after the Palaeocene/Eocene Thermal Maximum, a period of possibly widespread anoxia/dysoxia in deep-sea basins.

Antarctic Futures: An Assessment of Climate-Driven Changes in Ecosystem Structure, Function, and Service Provisioning in the Southern Ocean.

In this article, we analyze the impacts of climate change on Antarctic marine ecosystems. Observations demonstrate large-scale changes in the physical variables and circulation of the Southern Ocean driven by warming, stratospheric ozone depletion, and a positive Southern Annular Mode. Alterations in the physical environment are driving change through all levels of Antarctic marine food webs, which differ regionally. The distributions of key species, such as Antarctic krill, are also changing. Differential responses among predators reflect differences in species ecology. The impacts of climate change on Antarctic biodiversity will likely vary for different communities and depend on species range. Coastal communities and those of sub-Antarctic islands, especially range-restricted endemic communities, will likely suffer the greatest negative consequences of climate change. Simultaneously, ecosystem services in the Southern Ocean will likely increase. Such decoupling of ecosystem services and endemic species will require consideration in the management of human activities such as fishing in Antarctic marine ecosystems.

Defining the dopamine transporter proteome by convergent biochemical and in silico analyses.

Monoamine transporters play a key role in neuronal signaling by mediating reuptake of neurotransmitters from the synapse. The function of the dopamine transporter (DAT), an important member of this family of transporters, is regulated by multiple signaling mechanisms, which result in altered cell surface trafficking of DAT. Protein-protein interactions are likely critical for this mode of transporter regulation. In this study, we identified proteins associated with DAT by immunoprecipitation (IP) followed by mass spectrometry. We identified 20 proteins with diverse cellular functions that can be classified as trafficking proteins, cytoskeletal proteins, ion channels and extracellular matrix-associated proteins. DAT was found to associate with the voltage-gated potassium channel Kv2.1 and synapsin Ib, a protein involved in regulating neurotransmitter release. An in silico analysis provided evidence for common transcriptional regulation of the DAT proteome genes. In summary, this study identified a network of proteins that are primary candidates for functional regulation of the DAT, an important player in mechanisms of mental disorders and drug addiction.

ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation.

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.

Global biogeographic patterns in bipolar moss species.

A bipolar disjunction is an extreme, yet common, biogeographic pattern in non-vascular plants, yet its underlying mechanisms (vicariance or long-distance dispersal), origin and timing remain poorly understood. Here, combining a large-scale population dataset and multiple dating analyses, we examine the biogeography of four bipolar Polytrichales mosses, common to the Holarctic (temperate and polar Northern Hemisphere regions) and the Antarctic region (Antarctic, sub-Antarctic, southern South America) and other Southern Hemisphere (SH) regions. Our data reveal contrasting patterns, for three species were of Holarctic origin, with subsequent dispersal to the SH, while one, currently a particularly common species in the Holarctic (Polytrichum juniperinum), diversified in the Antarctic region and from here colonized both the Holarctic and other SH regions. Our findings suggest long-distance dispersal as the driver of bipolar disjunctions. We find such inter-hemispheric dispersals are rare, occurring on multi-million-year timescales. High-altitude tropical populations did not act as trans-equatorial 'stepping-stones', but rather were derived from later dispersal events. All arrivals to the Antarctic region occurred well before the Last Glacial Maximum and previous glaciations, suggesting that, despite the harsh climate during these past glacial maxima, plants have had a much longer presence in this southern region than previously thought.

Preparation, characterization and biological evaluation of cationic 99Tc/99mTc-dioxime complexes.

New cationic 99Tc/99Tc complexes formed with various symmetrical and asymmetrical vicinal dioximes of different carbon chain length (C5-C8) were synthesized by reduction of pertechnetate with BH4-, separated by HPLC and characterized by i.r./u.v./vis. spectroscopy, FAB mass spectrometry and electrophoresis. All complexes studied are trisdioximes containing boron as a constituent. Their lipophilicity, as assessed by the octanol/saline partition coefficient, ranges over almost four orders of magnitude. The myocardial uptake of the 99mTc complexes in mice proves to be lower than expected. The organ distributions are distinctly affected by the lipophilicity, the position of the dioxime group and the introduction of a terminal methoxy group.

Cenozoic climate change and diversification on the continental shelf and slope: evolution of gastropod diversity in the family Solariellidae (Trochoidea).

Recent expeditions have revealed high levels of biodiversity in the tropical deep-sea, yet little is known about the age or origin of this biodiversity, and large-scale molecular studies are still few in number. In this study, we had access to the largest number of solariellid gastropods ever collected for molecular studies, including many rare and unusual taxa. We used a Bayesian chronogram of these deep-sea gastropods (1) to test the hypothesis that deep-water communities arose onshore, (2) to determine whether Antarctica acted as a source of diversity for deep-water communities elsewhere and (3) to determine how factors like global climate change have affected evolution on the continental slope. We show that although fossil data suggest that solariellid gastropods likely arose in a shallow, tropical environment, interpretation of the molecular data is equivocal with respect to the origin of the group. On the other hand, the molecular data clearly show that Antarctic species sampled represent a recent invasion, rather than a relictual ancestral lineage. We also show that an abrupt period of global warming during the Palaeocene Eocene Thermal Maximum (PETM) leaves no molecular record of change in diversification rate in solariellids and that the group radiated before the PETM. Conversely, there is a substantial, although not significant increase in the rate of diversification of a major clade approximately 33.7 Mya, coinciding with a period of global cooling at the Eocene-Oligocene transition. Increased nutrients made available by contemporaneous changes to erosion, ocean circulation, tectonic events and upwelling may explain increased diversification, suggesting that food availability may have been a factor limiting exploitation of deep-sea habitats. Tectonic events that shaped diversification in reef-associated taxa and deep-water squat lobsters in central Indo-West Pacific were also probably important in the evolution of solariellids during the Oligo-Miocene.

The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

The biodiversity of the deep Southern Ocean benthos.

Our knowledge of the biodiversity of the Southern Ocean (SO) deep benthos is scarce. In this review, we describe the general biodiversity patterns of meio-, macro- and megafaunal taxa, based on historical and recent expeditions, and against the background of the geological events and phylogenetic relationships that have influenced the biodiversity and evolution of the investigated taxa. The relationship of the fauna to environmental parameters, such as water depth, sediment type, food availability and carbonate solubility, as well as species interrelationships, probably have shaped present-day biodiversity patterns as much as evolution. However, different taxa exhibit different large-scale biodiversity and biogeographic patterns. Moreover, there is rarely any clear relationship of biodiversity pattern with depth, latitude or environmental parameters, such as sediment composition or grain size. Similarities and differences between the SO biodiversity and biodiversity of global oceans are outlined. The high percentage (often more than 90%) of new species in almost all taxa, as well as the high degree of endemism of many groups, may reflect undersampling of the area, and it is likely to decrease as more information is gathered about SO deep-sea biodiversity by future expeditions. Indeed, among certain taxa such as the Foraminifera, close links at the species level are already apparent between deep Weddell Sea faunas and those from similar depths in the North Atlantic and Arctic. With regard to the vertical zonation from the shelf edge into deep water, biodiversity patterns among some taxa in the SO might differ from those in other deep-sea areas, due to the deep Antarctic shelf and the evolution of eurybathy in many species, as well as to deep-water production that can fuel the SO deep sea with freshly produced organic matter derived not only from phytoplankton, but also from ice algae.

PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile.

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.

The GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins.

The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.

Synthesis, characterization and biodistribution of the cationic technetium(II) 1,10-phenanthroline chelate.

[99Tc(phen)3]Cl2 was synthesized by reduction of pertechnetate with sodium borohydride in the presence of an excess of 1,10-phenanthroline and characterized by FAB mass spectrometry, magnetic susceptibility measurements, and i.r./vis/u.v. spectrophotometry. For biodistribution evaluation in mice [99mTc(phen)3]Cl2 could be obtained in a radiochemical purity of 92.5% using a similar procedure. The rather hydrophilic chelate was predominantly excreted by the kidneys. Its low blood retention resulted in a heart/blood activity ratio of 4.2 at 100 min post-injection.

Synthesis, characterization and evaluation of 99Tc/99mTc DIARS and DMPE complexes containing pentadecanoic acid.

The synthesis and purification of two new ligands, 15-[bis(3,4-dimethyl arsino)phenyl] pentadecanoic acid (FA-DIARS) and [bis(16,17-dimethyl phosphino)] heptadecanoic acid (FA-DMPE) and the preparation of their corresponding 99Tc/99mTc complexes of the general formula [Tc(III)L2Cl2]+, wherein L is FA-DIARS or FA-DMPE are reported. After careful purification by preparative HPLC, the 99Tc complexes were characterized by spectroscopy, while the 99mTc complexes were used to determine the biodistribution in mice. The results of the organ distribution studies indicated a relatively high uptake in heart (5-7% dose/g). The complexes were, however, found to be very slowly cleared from the blood, probably due to strong binding to serum proteins. Further, the heart to liver and heart to lung ratios were unfavourable. Attempts to improve the efficacy by synthesizing complexes containing one fatty acid per molecule are also described.

Type I brain hexokinase: axonal transport and membrane associations within central nervous system presynaptic terminals.

While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.

Isolation and characterization of Melanoplus sanguinipes adipokinetic hormone: a new member of the AKH/RPCH family.

A neuropeptide hormone isolated from corpora cardiaca of Melanoplus sanguinipes was purified by HPLC. The HPLC fractions were examined for adipokinetic activity with an in vivo bioassay. A single large UV absorbent peak was active in the mobilization of lipid while the other HPLC fractions showed no detectable activity. This large peak had a retention time and amino acid composition identical to synthetic Lom-AKH-I which was analyzed in a parallel manner. The primary sequence structure, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, was determined by automated gas-phase Edman degradation. The peptide was deblocked prior to sequencing using pyroglutamate aminopeptidase and the sequence was confirmed with mass spectrometry. The C-terminus of the peptide was determined to be blocked, as indicated by the lack of digestion with carboxypeptidase A. The knowledge of the primary sequence of Mes-AKH allows the use of a commercially available synthetic peptide and its antibodies for use in future research with Melanoplus sanguinipes.

Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.

Nebulin, a giant actin binding protein, coextends with actin and is thought to form a composite thin filament in the skeletal muscle sarcomere. To understand the molecular interactions between nebulin and actin, we have applied chemical cross-linking techniques to define molecular contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissociation constant of 4.9 microM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a zero-length cross-linker, l-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC), cross-linked the ND8-G-actin complex covalently without impairing actin's ability to polymerize. End-labeling Western blot and sequence and mass analyses of purified conjugated peptides revealed the cross-linking between lysine 5 of ND8 and the two N-terminal acidic residues of G-actin. Similarly, we have shown by end-labeling that cross-linking of ND8 to F-actin occurred at the N-terminus of actin protomer. The binding of nebulin to the N-terminus of actin is likely to be significant in its ability to affect actin polymerization. Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where Sl, tropomyosin, and several actin binding proteins are known to interact.

Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma.

Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a nonamyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

Stoichiometry of subunits and heme content of hemoglobin from the earthworm Lumbricus terrestris.

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, c (forming a disulfide-linked trimer), and d ("monomer"). Additional structural chains, "linkers," are required for the assembly of the approximately 200-polypeptide molecule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densitometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3% linkers on the basis of both 220-nm absorbance and amino acid analysis (Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C., Chait, B. C., and Riggs, A. F. (1993) J. Biol. Chem. 268, 13539-13547). The subunit proportions have now been redetermined by SDS capillary electrophoresis as a test of the HPLC results. The electrophoresis, monitored at 214 nm, avoided the use of Coomassie Blue and provided results identical with those obtained by HPLC. Capillary electrophoresis monitored at both 214 and 415 nm was used to show that linker chains do not bind heme. Heme content has been found to be 2.9% by determination of hemin, amino acid analysis and dry weight. Measurement of the rate of hemin loss from oxidized L. terrestris Hb shows that high rates of loss can account for values of heme content significantly below 2.9% (or 0.26% iron).