Poster J13 - Analysis of Metal-Protein Complexes Using Colorimetric Assays

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Elizabeth Willenborg

Undergraduate Student
West Virginia University
Morgantown, West Virginia, United States

Co-Author(s)

GP

Gia Huy Pham

Graduate Student
West Virginia University
Bruceton Mills, West Virginia, United States

Primary Investigator(s)

CD

Cerasela Zoica Dinu

Professor
Chemical and Biomedical Engineering Department, West Virginia University, United States

Introduction: Bicinchoninic Acid Assay of BCA is a colorimetric technique used to quantify the amount of protein in a sample. The assay relies on a biuret reaction under alkaline conditions in which peptide and amino acids such as cysteine, tryptophan and tyrosine reduce cupric (Cu2+) to cuprous ion (Cu+). The bicinchoninic acid cuprous complex forms a purple color read at 562 nm using spectroscopy where the intensity of the color is in direct relation to the concentration of protein present in solution. BCA has applications in biochemical, biomedical, and biotechnology research, as well as in clinical diagnostics. We hypothesized that BCA analysis could be used to evaluate the formation of new metal-enzyme complexes, namely Rubisco-Mg-based enzyme complexes. Moreover, BCA analysis is the first required step to be employed to test such complex functionality. Rubisco, a primary carboxylase of the photosynthetic process is widely abundant for human consumption. Complexes-based on Rubisco were of interest for this work due to their envisioned ability to serve as green sources for CO2 entrapment and consumption in synthetic environment. Such a strategy is thus of interest to reduce challenges associated with escalating global climate changes due to greenhouse has emission. We further anticipate that the work performed herein provides a viable evaluation platform towards demonstrations on how an enzyme-based complex could be used for CO2 capture.

Materials and

Methods: Mg/RuBisCO complexes were created at room temperature and under different incubation times (i.e., 1, 3, 6, 12, and 24 hours) and ratios (i.e., 50:1 and 100:1 Mg:RuBisCO) respectively (Figure 1). The complex was first tested with BCA assay. Briefly, 50 parts of reagent A was combined with 1 part of reagent B to create a working reagent. The complex’s supernatant underwent a passive-transport dialysis for 4 hours with a selective permeable membrane tube (MWCO = 30k) and with K-PBS pH 7.8 as the dialysate to filter out any interference ions that could ultimately produce false signals. The BCA working reagent combined with 50µL of the corresponding Mg/RuBisCO complex and was then incubated at 37°C for 30 minutes. The quantification was based on the solution changing the color from light green to purple. The intensity of this mixture post-incubation was observed at 562nm. The control BCA assay was performed with RuBisCO to set a calibration curve, and the quantity of enzyme in the metal-enzyme complexes was compared against the control calibration curve.

Results, Conclusions, and Discussions: Our analysis showed that when BCA was used to evaluate the amount of enzyme in the complex, a time and ratio-dependent colorimetric distribution was observed. Further, our analysis showed that dialysis post-synthesis for 4 h was crucial to determine the quantity of RuBisCO in the Mg/RuBisCO complex. In particular, prior to dialysis, the supernatant of the complex had shown precipitation upon reacting with the copper ions presented in the BCA assay (Figure 3a). Upon evaluating this precipitate with scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM/EDX; Figure 3b), the precipitate presented a high concentration of oxygen, which was then interpreted as the supernatant contained an oxide-based or a hydroxide-based that could ultimately precipitated with the copper presented in the BCA assay reagents (Figure 3c).
The BCA assay-based results demonstrated the simplicity and reproducibility of our approach when used in determining the quantity of enzyme in the enzyme-based complex analysis. Specifically, the quantity of RuBisCO in the Mg/RuBisCO complex was about 0.45 mg/mL and 0.60 mg/mL for 50:1 and 100:1 Mg:RuBisCO from 6 h to 24 h, respectively. In translation, this plateau showed that the maximum binding between Mg2+ and RuBisCO could sustain at ~45% and ~60% of the original starting amount of RuBisCO for 50:1 and 100:1, respectively, from 6 h, of which inferred that the amount of Mg played a crucial role of binding efficiency with RuBisCO in the Mg/RuBisCO complex. Further analysis needs to be conducted to determine the functionality of the enzyme in the complex and understand how such metal-protein complex could be employed for CO2 mitigation.

References:
1. Quantification and analysis of proteins, Chang-Hui Shen, in Diagnostic Molecular Biology (Second Edition), 2023
2. Rational Design of Enzyme-Nanomaterials, B.A. Otieno, ... J.F. Rusling, in Methods in Enzymology, 2016
3.Enzymes of Energy Technology, David A. Collins, Marina G. Kalyuzhnaya, in Methods in Enzymology, 2018
4.Active nano interfaces based on enzyme Carbonic Anhydrase and Metal-Organic Framework for carbon dioxide reduction, Liu, Q., Bau, X., Pham, H., Hu, J., Dinu, C.Z., Nanomaterials, 11(4): 1008. (2021).

Acknowledgements (Optional):