Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG

Abstract

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.•This method enables the combined measurement of bilirubin and biliverdin in the nM range.•The method does not require solvent extraction or protein precipitation of the samples.

Keywords: Assay optimal parameters; Biliverdin; Biological fluids; Combined biliverdin and bilirubin analysis with HUG; Plasma.

Graphical abstract

Scheme 1

Typical 96-well calibration plate. The plate is loaded with BV and BR standard solutions. The remaining wells can be loaded with real samples. If you need to load a larger number of samples, it is not necessary to introduce a calibration curve for each plate.

Fig. 1

Progress of the BVR reaction with either NADH or NADPH. The variation of the angular coefficient (obtained plotting the BR and BV concentrations vs fluorescence) over time at 25 °C is shown at pH 7.4 (empty symbols) and pH 8.5 (solid symbols) by using 10 µM NADPH (circles) or NADH (squares). Data were fitted to the equation: $y=y_M(1-e^{-kx})$, where y_M is the maximum reached value (697 ± 20$)$, k is the rate constant (min^-1): 0.09546 with NADPH at pH 7.4; 0.1095 with NADPH at pH 8.5; 0.007551 with NADH at pH 7.4. Data obtained with NADH at pH 8.5 were fitted to the equation y = ax + c, where a = 0.4513, c = −11,53. Some error bars are shorter than the size of the symbol.

Fig. 2

The dependence of the BVR reaction on the coenzyme concentration. Variation of angular coefficient (obtained plotting the BV concentrations vs fluorescence) as a function of increasing coenzyme concentrations. The reaction took place in the presence of increasing concentration (0–100 µM) of NADPH at pH 8.5 (solid symbols) or NADH at pH 7.4 (open symbols) using 0.1875 IU/mL BVR. Data were fitted to the equation y = ax/k + x where a is the highest angular coefficient obtained in these experimental conditions, k is the concentration of coenzyme that gave half maximal angular coefficient. The parameters obtained at 25 °C were k = 0.0760 and a = 735.1 for NADPH (dotted line); k = 2.854 and a = 775.7 for NADH (continuous line).

Fig. 3

The dependence of the BVR reaction on the temperature. The variation of the angular coefficient in nM^-1 (obtained plotting the BV concentrations vs fluorescence) over time is represented at 25 °C (solid symbols) and at 37 °C (open symbols). Data were fitted to the equation: $y=y_M(1-e^{-kx})$ where y_M is the maximum reached value, k is the rate constant (min^-1); k = 0.158 at 25 °C; k = 0.189 at 37 °C with 0.1 mM NADPH (circles and continuous line); k = 0.00652 at 25 °C; k = 0.00977 at 37 °C with 0.1 mM NADH (squares and dotted line).

Fig. 4

The dependence of the BVR reaction on the enzyme concentration. (A) kinetics of angular coefficient variation at different BVR concentrations. After 16 h of incubation, data show that the optimal BVR concentration was at least 0.185 U/mL. Data were fitted to the equation: $y=y_M(1-e^{-kx})$, where y_M is the maximum reached value; k is the rate constant (h^-1); (B) the rate constant of reaction at each BVR concentration.

Fig. 5

Analysis of both bilirubin and biliverdin by HUG. Analysis of BV and BR was performed as described in the text. Fluorescence data from individual calibration tests (n = 19 with BV, green circles; n = 18 with BR, orange circles) were combined and fitted to the linear equation y = a + b*x. The parameters obtained were: a = 305 ± 177; b = 748 ± 9; R² = 0.97.