A spectrophotometric method for estimating hemin in biological systems.

Hemin chlorides exhibit two absorption maxima in the Soret region, one at about 360-380 nm (S' band) and the other between 400 and 430 nm (S band). We present here a simple and fast spectrophotometric assay to determine concentration of hemin between 1.15 and 9.20 microM employing the Soret region (S' band) as a reference. In this method the hemin is quantitatively extracted from biological materials by acidified chloroform. By recording the absorbance of the chloroform extract at its maximum peak at 388, 450, and 330 nm and applying the correction formula A(c)=2A388-(A450+A330), a very good linear correlation between the A(c) and the concentration of hemin is attained. The method can be used to estimate hemin in the presence of protein (0.06-5.00 mg/ml) and porphyrin (0.19-2.97 microM). Compared with the pyridine hemochromogen method, the assay reported here is highly reproducible, with 15- to 30-fold more sensitivity, and it allows the quantification of four times lower hemin concentrations.

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi.

BACKGROUND AND AIMS: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. METHODS: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. RESULTS: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13+/-0.49EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. CONCLUSIONS: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.

Saccharomyces Cerevisae: actividad de porfobilinogenasa en una cepa salvaje y su mutante hemo-deficiente / Saccharomyces cerevisiae: porphobilinogenase activity in a wild strain and its haem mutant

Se realizaron las curvas de crecimiento para las cepas D273-10B y B231 de Saccharomyces cerevisiae, obteniéndose los tiempos de generación (G = 1,26 y 3,20 horas respectivamente). Se han establecido las conclusiones óptimas para la ruptura celular y extracción de la Porfobilinogenasa (PBGasa). Se han determinado la composición del sistema, tiempo, pH y atmósfera de incubación óptima para la medición de la actividad de PBGasa en ambas cepas. Se midieron las constantes cinéticas, obteniéndose Km del mismo orden para ambas cepas de Saccharomyces cerevisiae, mientras que la velocidad máxima resultó ser para la cepa mutante el doble del valor correspondiente a la salvaje. Se estudió la variación de la actividad enzimática en función del tiempo de crecimiento, hallándose un comportamiento significativamente diferente entre ambas cepas. Para la mutante se apreció un máximo bien definido entre las 20 y 25 h. y además con una actividad de aproximadamente 2 veces del valor medido para la cepa salvaje en iguales condiciones