Alignment of sequences of vertebrate 405 , and Glu 457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate nonconserved in the overall family with alanines by sitedirected mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu 405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V max . The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu 405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase. ␤-Carotene 15,15Ј-monooxygenase-1 (BCMO1)1 is the initial enzyme step in the biosynthesis of vitamin A in animals, symmetrically cleaving ␤-carotene to produce two molecules of alltrans-retinal (1). In the last several years, a number of BCMO1 proteins have been cloned and biochemically characterized (2-7). BCMO1 belongs to a family of oxygenases of diverse activities, including lignostilbene dioxygenase in bacteria (8 -10); an epoxycarotenoid-cleaving enzyme required for abscisic acid biosynthesis in plants (11); and mammalian RPE65, a retinal pigment epithelial protein required for the production of the visual chromophore 11-cis-retinal in the visual cycle (12). The most recent addition to the characterized carotenoid oxygenases is cyanobacterial apocarotenoid 15,15Ј-oxygenase PCC 6803 (ACO) (13). Despite their unique functionalities and the obvious importance of these proteins, structural studies on this family have been hindered by technical difficulties, and only recently has the crystal structure of eubacterial ACO been published (14).Originally, Drosophila BCMO1 was identified by homology to a previously characterized plant 9-cis-epoxycarotenoid dioxygenase (15) and by its ability to cleave ␤-carotene (4). Subsequently, mouse BCMO1 was identified by our laboratory based on its homology to mammalian RPE65 (3). Because the biochemical function and mechanism of BCMO1 have been studied in detail (3, 5, 7), it is functionally the best characterized mammalian member of the family and could serve as a model for studying putative vertebrate oxygenases of more elusive function such as RPE65 and BCMO2 (16).Although there is a weak overall identity (Ͻ10%) am...

The design and implementation of two different interfaces for capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) are described. These interfaces will allow for on-line analysis of CE effluents with ICP-MS detection. One interface is based on a concentric tube nebulizer and the other on a standard cross-flow nebulizer. These systems were investigated in parallel and their performances, under various experimental conditions, were compared. Each interface possesses a unique set of advantages and shortcomings. Recognizing that typical sample flow rates for ICP-MS are of the order of ml min 21 and that the flow rates for CE are a few nl min 21 , some difficulties in flow compatibility are encountered. Aspects discussed include interface considerations, flow compatibility and the influence of flow rates on the overall sensitivity. Several guidelines are provided for workers interested in implementing a CE-ICP-MS instrument for elemental speciation. The Cd detection limits in rabbit metallothionein were 2.36 and 0.21 mg ml 21 for the concentric and cross-flow nebulizers, respectively.

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Simultaneous multi-element graphite furnace atomic absorption spectrometric analysis of solids prepared as slurries is investigated. Slurries were prepared using 5-10 mg of sample and 5 ml of dilute nitric acid. Analyses were performed on a simultaneous multi-element atomic absorption spectrometer with a continuum source (SIMAAC) using platform atomisation, integrated absorbance measurements, 20-kl sample volumes and calibration standards prepared in 5% HN03. As many as seven elements were determined simultaneously in a total of ten different National Bureau of Standards (NBS) reference materials representing a wide variety of sample matrices. Ultrasonic agitation of the slurry preparations proved useful in avoiding settling of the particles. Typically, average accuracies of 100 k 12% were observed.

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A standard cross-flow nebulizer and commercially available microconcentric nebulizer (MCN-100) have been compared for capillary zone electrophoresis (CZE) inductively coupled plasma mass spectrometry (ICP-MS) measurements. Metallothionein samples were separated and detected to characterize the performance of the two nebulizers for chemical speciation measurements. The MCN-100 offered improved sensitivity and lower detection limits compared to the cross-flow nebulizer, but provided slightly poorer resolution. The detection limit for 114Cd in metallothionein solutions was 90 ng/g with the cross-flow nebulizer and 10 ng/g with the MCN-100 for ∼ 4 nL injections. These values correspond to absolute detection limits of 360 fg Cd in the injected sample with the cross-flow nebulizer and 40 fg Cd for the MCN-100. Quantitation of Cd in metallothioneins (rabbit liver and horse kidney) with the use of a well-characterized rabbit liver metallothionein sample as the calibration standard is reported. Measured Cd concentrations agreed with results obtained by both graphite furnace atomic absorption spectroscopy (AAS) and solution nebulization ICP-MS.

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