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Selected Publication:

Type of publication: Baccalaureate Thesis
Type of document:

Year: 2015

Authors: Tonner, Matthias

Title: Adaption of photospectroscopic assays to determine activities of enzymes of the heme degradation pathway in small sample volumes.

Other title: Anpassung der photospektroskopischen Bestimmung der Enzyme des Häm- abbauenden Wegs in kleinen Probenvolumina

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 39.


Duvigneau Catharina

Staniek Katrin

Vetmed Research Units:
Institute for Medical Biochemistry

Graduation date: 18.12.15

The heme degradation pathway converts heme to bilirubin (BR), carbon monoxide and ferric ion. The two NADPH dependent enzymes which are responsible for the conversion are heme oxygenase (HO), which converts heme to biliverdin (BV), and biliverdin reductase (BVR), which converts BV to BR. HO forms the rate limiting step of the degradation and plays an important role for oxidative stress an cell homeostasis. BVR plays also an important role in pathologic conditions involving biases of the redox balance. Therefore it is frequently necessary to determine changes of the activity of the heme degradation pathway. The biochemical enzymatic assay used previously is based on the original coupled enzymatic assay described by Tenhunen et al. (1968) subsequently followed by an extraction of the formed BR with an organic solvent and a subsequently spectrophotometric measurement to determine the concentration. This assay works well for tissues with high enzymatic activity but for tissues with low activity or small sample volumes the formed amount of BR is not sufficient to allow precise determination of BR concentration and may fall below the limit of detection for the spectrophotometric measurement. Therefore, the aim of this study was to optimize the HO assay for small sample volumes. During the optimization we had to solve several problems: First the high protein content of the samples interfered with the extractability of BR, second the original extracting solvent chloroform was not viable for very low BR concentrations. We solved the protein problem by adding caffeine solution prior to the extraction, which almost negated the protein effect and enhanced the BR yield by factor 2. The problem of the extracting solvent was solved by substituting chloroform with benzene. Doing that allowed us to halve the overall assay volume, which further increased the sensitivity. By comparing the classic protocol with the optimized protocol we have shown that the yield of the assay normalized for protein is enhanced by a factor from 3,5 to 4,6 depending on the enzymatic activity of the investigated tissue.

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