ALBERT

Notes: Summary A phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local “molecular clock” calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Notes: Abstract Artemia has evolved the longest known concatenation of hemoglobin domains, the α subunit containing nine domains and the β subunit having a similar size. Translation of the cDNA sequence of the α subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit. The interdomain linker sequences were 14%–64% identical (62%–91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule. The position of interdomain introns and the character of a variable residue early in the linker are compatible with the nine-domain polymer having evolved through gene duplication reflected in globin domain fusion incorporating an extension specifically of the N-terminus. The multiplication of an original single-domain globin gene to give the present nine is estimated from sequence differences, allowing for multiple mutations at individual sites, to have occurred in a period at least 500–700 million years ago.

Notes: The nemertean worm Cerebratulus lacteus neural tissue haemoglobin (109 amino acids, the shortest known haemoglobin) has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant native data set has been collected at the Cu Kα wavelength to 2.05 Å resolution. The crystals belong to the orthorhombic P212121 space group, with unit-cell parameters a = 42.5, b = 43.1, c = 60.2 Å and one molecule per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe atom, thus paving the way for MAD/SAD structure determination.

Notes: [Auszug] The globins are proteins famed for their oxygen-carrying capacity. Two types are known in vertebrates: haemoglobin, found in the blood, and myoglobin, located in muscle. Now a third has been identified, as described by Burmester et al. on page 520 of this issue. It is found ...

Notes: Abstract Inductively coupled plasma mass spectrometry of environmental and biological samples is often hampered by spectral and non-spectral interferences. Spectral interferences, caused by the limited resolution of the quadrupole mass spectrometer, can be eliminated in a variety of ways. For their identification inspection of a signal versus carrier gas flowrate is useful. Anion exchange allows the removal of most S and Cl containing compounds, which are at the origin of the majority of spectral interferences. Matrix modification, for example the addition of ethanol and subsequent optimization of the gas flow rates in a number of cases enables the reduction of the interferences to insignificant values. Often a mathematical correction based on isotopic signal ratios can be applied. Non-spectral interferences can be divided in reversible, that is occurring while the sample is being measured, and irreversible matrix effects, that is clogging of the nebulizer and sampling orifices or deposition on the torch or in the ion lens stack. The errors associated with non-spectral interferences can be eliminated by appropriate calibration procedures, adapted sample preparation or limitation of the amount of sample delivered to nebulizer, plasma and sampling devices, for example by the application of flow injection. Applications of all the elimination procedures are described for the analysis of sea-water, estuarine water, soil and sewage extracts, percolate water, urine, serum and wine.

Notes: Abstract The importance of mass spectrometry for the analysis of biological material is illustrated by reviewing the different mass spectrometric methods applied and describing some typical applications published recently. Though atomic absorption spectrometry is used in the majority of analyses of biological material, most mass spectrometric methods have been used to some extent for trace element determination in biomedical research. The relative importance of the different methods is estimated by reviewing recent research papers. It is striking that especially inductively coupled plasma mass spectrometry is increasingly being applied, partly because the method can be used on-line after chromatographic separation, in speciation studies. Mass spectrometric methods prove to offer unique possibilities in stable isotope tracer studies and for this purpose also experimentally demanding methods such as thermal ionization mass spectrometry and accelerator mass spectrometry are frequently used.

Notes: Abstract During the past decade, inductively coupled plasma mass spectrometry (ICPMS) has evolved from a delicate research tool, intended for the well-trained scientist only, into a more robust and well-established analytical technique for trace and ultra-trace element determination, with a few thousand of instruments used worldwide. Despite this immense success, it should be realized that in its ’standard configuration’– i.e. equipped with a pneumatic nebulizer for sample introduction and with a quadrupole filter – ICPMS also shows a number of important limitations and disadvantages: (i) the occurrence of spectral interferences may hamper accurate trace element determination, (ii) solid samples have to be taken into solution prior to analysis and (iii) no information on the ‘chemical form’ in which an element appears can be obtained. Self-evidently, efforts have been and still are made to overcome the aforementioned limitations to the largest possible extent. The application of a double focusing sector field mass spectrometer in ICPMS instrumentation offers a higher mass resolution, such that spectral overlap can be avoided to an important extent. Additionally, in a sector field instrument, photons are efficiently eliminated from the ion beam, resulting in very low background intensities, making it also very well-suited for extreme trace analysis. Also the combination of the ICP as an ion source and a quadrupole filter operated in a so-called ‘alternate’ stability region, an ion trap or a Fourier transform ion cyclotron resonance mass spectrometer allows high(er) mass resolution to be obtained. With modern quadrupole-based instruments, important types of spectral interferences can be avoided by working under ‘cool plasma’ conditions or by applying a collision cell. The use of electrothermal vaporization (ETV) or especially laser ablation (LA) for sample introduction permits direct analysis of solid samples with sufficient accuracy for many purposes. The application range of LA-ICPMS has become very wide and the introduction of UV lasers has led to an improved spatial resolution. Solid sampling ETV-ICPMS on the other hand can be used for some specific applications only, but accurate calibration is more straightforward than with LA-ICPMS. Limited multi-element capabilities, resulting from the transient signals observed with ETV or single shot LA, can be avoided by the use of a time-of-flight (TOF) ICPMS instrument. Finally, when combined with a powerful chromatographic separation technique, an ICP-mass spectrometer can be used as a highly sensitive, element-specific multi-element detector in elemental speciation studies. Especially liquid (HPLC-ICPMS) and – to a lesser extent – gas (GC-ICPMS) chromatography have already been widely used in combination with ICPMS. In speciation work, sample preparation is often observed to be troublesome and this aspect is presently receiving considerable attention. For GC-ICPMS, new sample pretreatment approaches, such as headspace solid phase microextraction (headspace SPME) and the purge-and-trap technique have been introduced. Also supercritical fluid chromatography (SFC) and capillary electrophoresis (CE) show potential to be of use in combination with ICPMS, but so far the application ranges of SFC-ICPMS and CE-ICPMS are rather limited. It is the aim of the present paper to concisely discuss the aforementioned recent ’trends’ in ICPMS, using selected real-life applications reported in the literature.