Maurice V. Laycock

Analysis of paralytic shellfish poisons by capillary electrophoresis

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.

Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus)

Abstract Three clones were isolated from a lobster digestive gland cDNA library, using oligonucleotide probes based on the partial amino terminal sequence of a digestive cysteine proteinase. The cDNAs, LCP1, LCP2 and LCP3 encode preproenzymes of 322, 323 and 321 amino acid residues, and putative mature enzymes of 217, 216 and 215 residues, respectively. Calculated mature protein molecular masses are 23386 (LCP1), 23093 (LCP2) and 23255 (LCP3) Sequence alignments show that the lobster enzymes are more similar to L (55–62% identity) than H (42–44%) or B (22–24%) cathepsins. Southern analysis indicated as many as eleven genes related to the three cDNAs.Southern analysis indicated as many as eleven genes related to the three cDNAs.

Sensitivity of lateral flow tests to mixtures of saxitoxins and applications to shellfish and phytoplankton monitoring

We have investigated some characteristics of antibodies in the lateral flow format for detecting paralytic shellfish poisoning (PSP) toxins and compared them with the mouse bioassay (MBA). The MBA is still the most reliable test for toxicity in shellfish because it provides an estimate of toxicity directly and can include more than one contaminant. Most other methods, including those involving antibodies, provide estimates of toxin concentration from which toxicity is implied or calculated using conversion factors. Antibody methods suffer from an additional deficiency as sensitivities to the different PSP analogues are unequal. Furthermore, these differences in cross-reactivity are unrelated to differences in specific toxicities. We have addressed the question of what is the toxicity of a sample at the limit of detection (LOD) of the Jellett Rapid Testing Ltd (JRT) lateral flow immunochromatographic (LFI) test. A way to calculate sensitivity to toxicity from toxin profiles is presented and used to examine a variety of PSP toxin mixtures. The calculated values for the sensitivity of the JRT (toxicity at the LOD) for separate PSP toxin analogues may vary over a wide range, but for complex mixtures, typical of natural samples, the range is much narrower. An analysis of PSP toxin profiles of 339 samples from Alaska, Britain, Canada (BC), and USA (Maine) shows the distribution of calculated toxicities at the LOD. The majority (76%) falls within the range 20-50 microg STX eq/100g with a mean at 32 microgSTXeq/100g which is similar to that of the MBA. Observed data from independent parallel studies with the JRT and MBA with a total of 3492 samples from regulatory laboratories in different countries in the period 2003-2007 show close agreement between the two methods. All samples that were found to be positive with the MBA were also positive with the JRT except for one which indicated a false negative rate of less than 0.03% of all samples tested. The JRT for PSP was designed to be more sensitive than the MBA in order to be used as a screen to reduce the high proportion of negative samples encountered in routine monitoring. Toxicity at the LOD varies depending on the mixture of PSP toxins and false positives are therefore inevitable. In this study false positives accounted for between 1.4% and 55% of the total number of samples tested. This would also depend on whether contamination was endemic or rare in the different locations. The data from regulatory monitoring for PSP show that in all areas the majority of samples are negative and so the use of a screen would result in a significant reduction in the use of mice.

Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins

A sample stacking procedure is presented for the capillary electrophoretic (CE) separation of paralytic shellfish poisoning (PSP) toxins dissolved in high ionic strength buffers. The application of such a stacking procedure prior to the zone electrophoretic separation is demonstrated for the analysis of decarbamoyl toxins arising from the digestion of PSP toxins by an hydrolytic enzyme from little neck clams (Protothaca staminea). Improvements in separation efficiency facilitated identification and quantitation of substrates and enzymatic products present in the digest using CE. The separation conditions developed were found to be entirely compatible with electrospray mass spectrometry, which permitted the analysis of PSP toxins and their decarbamoyl derivatives present in the low micromolar range in crude enzyme digests. The products released during the enzymatic digestion were identified using CE combined with tandem mass spectrometry.

The biosynthesis of phycocyanobilin in Anacystis nidulans

Abstract Aminolevulinic acid, the common precursor of linear and cyclic tetrapyrroles, can arise either by condensation of succinate and glycine or from the ent

Characterization of a mixture of lobster digestive cysteine proteinases by ionspray mass spectrometry and tryptic mapping with LC—MS and LC—MS—MS☆

Abstract An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography—mass spectrometry (LC—MS) and LC—MS—MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC—MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC—MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray ® LC—MS interface (pneumatically assisted electrospray).

A simple procedure for sulfation and 35S radiolabelling of paralytic shellfish poisoning (PSP) gonyautoxins

A method is described to sulfate PSP toxins at various positions in the molecule and to prepare 35S labelled compounds using H2(35)SO4 in the presence of dicyclohexylcarbodiimide (DCC). The 11-sulfates of saxitoxin and neosaxitoxin, known as gonyautoxins, are often the most abundant of the PSP toxins in algae and contaminated shellfish. Receptor site binding and antibody assays based on these analogues should, therefore, better reflect toxicity than those in which saxitoxin is used. Although the specific activity of 35S-gonyautoxins is lower than that of commercially available 3H-saxitoxin, the label is strongly bound and is not lost through proton exchange with water as occurs with tritiated saxitoxin. The labelling procedure is rapid, inexpensive and can be done on a small scale. Sulfate can be removed from the 11-position of GTXs in methanolic-HCl and from the 21-position by mild acid hydrolysis and H2(35)SO4 added in 5-10-fold excess. Addition or exchange occurs rapidly on mixing DCC in dimethylformamide with dry toxin and sulfate. Reaction conditions were optimized and reaction products identified by capillary electrophoresis, autoradiography and ionspray mass spectrometry. Together with methods for selective removal of sulfate, the sulfation reaction provides an additional way to prepare some of the naturally occurring derivatives of saxitoxin, many of which are sulfates.