Determination of Sulfhydryl Groups in Certain Biological Substances

Chinard, Francis P.; Hellerman, Leslie

doi:10.1002/9780470110171.ch1

Cited by 56 publications

(1 citation statement)

References 113 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The reaction was maintained at pH 7–7.5 with the NaHCO 3 solution. Succinyl CoA formation was checked by the hydroxamic acid method (disappearance of succinic anhydride) of Lippman and Tuttle [22] and the nitroprusside method for acyl mercaptans of Cinnard and Hellerman [23]. …”

Section: Methodsmentioning

confidence: 99%

A method for determining δ-aminolevulinic acid synthase activity in homogenized cells and tissues

Bergonia¹,

Franklin²,

Kushner³

et al. 2015

Clinical Biochemistry

View full text Add to dashboard Cite

Objective In mammalian cells the rate-limiting step in heme biosynthesis is the formation of δ-aminolevulinic acid (ALA). The reaction intermediates, porphyrins and iron and the final product, heme can be highly cytotoxic if allowed to accumulate. The importance of maintaining the levels of metabolic intermediates and heme within a narrow range is apparent based on the complex homeostatic system(s) that have developed. Ultimately, determining the enzymatic activity of ALA synthase (ALAS) present in the mitochondria is highly beneficial to confirm the effects of the transcriptional, translational and post-translational events. The aim of this study was to develop a highly sensitive assay for ALAS that could be used on whole tissue or cellular homogenates. Design and methods A systematic approach was used to optimize steps in formation of ALA by ALAS. Reducing the signal to noise ratio for the assay was achieved by derivatizing the ALA formed into a fluorescent product that could be efficiently separated by ultra performance liquid chromatography (UPLC) from other derivatized primary amines. The stability of whole tissue homogenate and cellular homogenate was determined after extended storage at −80°C. Conclusions A method for assaying ALAS has been developed that can be used with tissue homogenates or cellular lysates. There is no need to purify mitochondria and radiolabeled substrates are not needed for this assay. General laboratory reagents can be used to prepare the samples. Standard UPLC chromatography will resolve the derivatized ALA peak. Samples of tissue homogenate can be stored for approximately one year without significant loss of enzymatic activity.

show abstract

Section: Methodsmentioning

confidence: 99%