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Because mycotoxins occur worldwide in grain and grain products, evaluating their effects on the health of the population has become important. The development of a high performance liquid chromatographic (HPLC) procedure was investigated for the analysis of aflatoxin B1, citrinin, and ochratoxin A, from hydrolyzed human urine. Solid-phase extraction (SPE) was used for sample clean-up and concentration. Reversed-phase liquid chromatography (RPLC) was used with fluorescence detection for sample analysis. The presence of aflatoxin B1 was confirmed by converting it to the hemiacetal. With 10 mL of urine, the detection limit for aflatoxin B1 and ochratoxin A was in the high parts per trillion (ppt) and that for citrinin was about 10 ppb.

An equilibrium gel permeation technique has been developed for determining as a function of oxygenation state the equilibrium constants for association of hemoglobin subunits. By using this method, the dimer-tetramer constant for human hemoglobin at a partial oxygenation state corresponding to 20% saturation for tetramers has been determined as 3.7 X 106 M-1 (dimers). Under the same conditions the corresponding constant for fully oxygenated hemoglobin is 4.1 X 10&M-1. These results are found to be in good agreement with the predicted behavior of the association reaction based upon oxygen binding curves measured as a function of protein concentration. Thus a high degree of consistency is found between the two independent experimental approaches to the reciprocal effects of this linkage system, lending support to the theory proposed earlier for these phenomena.

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Carbamoylphosphate synthetase from Pseudomonas ueruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122000 and 44000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine ( K , 0.15 mM) or NH3 (K, 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aerugino.ra does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.Carbamoylphosphate is an intermediate in the biosynthesis of arginine and pyrimidines. In enteric bacteria [l -51 it is synthesized by a single enzyme, carbamoylphosphate synthetase, which catalyzes the following reaction :Ammonia can replace glutamine as a nitrogen donor in the reaction but the affinity for ammonia is much less than that for glutamine; the latter substrate is considered the physiological nitrogen donor. The enteric enzymes [l -51 are all subject to cumulative repression by arginine and a pyrimidine compound and to feedback control: U M P inhibits activity and ornithine stimulates it. The combined effects provide an efficient mechanism for controlling the supply of carbamoylphosphate for arginine and pyrimidine biosynthesis.Examination of carbamoylphosphate metabolism in Pseudomonas aeruginosa is of particular interest in view of the significant differences that distinguish arginine metabolism in this organism from that in enteric bacteria [6,7]. These differences include the ability of P. ueruginosu and other fluorescent pseudomonads to degrade arginine via the arginine deiminase pathway (Fig. 1). This pathway involves the activity of carbamate kinase which catalyzes the phosphorylation of ADP by carbamoylphosphate [7] according to the following reaction :The possible presence of two enzyme...