Introduction:
We all know that there are many biochemical reactions taking place in living cells which require thermodynamically favorable conditions. The cytoplasm of these living cells is crowded by biomolecules. To understand these biochemical reactions and the factors affecting them, standard molecular crowder systems, like PEG, dextrans, etc. are used to mimic the crowded environment of living cells. However, it was observed that PEG 400 leads to the complexation of cations in non-aqueous solutions below a particular equilibrium association constant. Now, the question arises whether the high concentration of these crowders can affect the ionic strength of the buffers and thus, the equilibrium constant of biochemical complex formations? Let’s seek the answer to this question.
A recent research conducted by the team of professor Robert Holyst at the Institute of Physical Chemistry of the Polish Academy of Sciences shed light on this topic. In their research article titled “Ion Complexation Explains Orders of Magnitude Changes in the Equilibrium Constant of Biochemical Reactions in Buffers Crowded by Nonionic Compounds”, they investigated the effect of crowded environment and ionic strength on the non-covalent complex formation.
Research:
The effect of molecular crowding was studied using hybridization of complementary DNA oligonucleotides as a model biochemical reaction at the nanomolar concentration range because this process is sensitive to the ionic strength. Thus, it will act as a good indicator of complexation of ions by the crowding polymers.
Firstly, the effect of molecular crowding on DNA hybridization was demonstrated by determining the equilibrium constant (K) using the brightness analysis method. It was observed that the change in K value was negligible at low concentrations of the crowders, but for high concentrations, it decreases by 2−3 orders of magnitude because of the complexation of sodium cations by weak interaction with crowder molecules which leads to the ion deficiency. The effect of ionic strength on DNA hybridization was then demonstrated. It was observed that the increase in ionic strength leads to increase in interactions between substrates, but an increase above the certain value results in the reducing effect on the bound fraction. Lastly, potentiometric measurements using an ion-selective electrode was used to determine the complexation of sodium ions by crowders which was confirmed by retrieving the original K value of buffer by adding a calculated amount of ions to compensate the loss by complexation. Finally, it was proposed that the complexation of the ions by the crowders changes the equilibrium constant of the reaction, and not the alteration in volume available to reactants by the crowders.
Conclusion:
To conclude, researchers experimentally showed that the complexation of cations from the buffer by non-ionic crowding polymers may be responsible for the major decrease in equilibrium constant of the biochemical reactions, and not the molecular crowding, as it was previously believed. This research will prove beneficial for the further studies to obtain precise and accurate results in the in vitro experiments.
Reference:
Krzysztof Bielec et al, Ion Complexation Explains Orders of Magnitude Changes in the Equilibrium Constant of Biochemical Reactions in Buffers Crowded by Nonionic Compounds, The Journal of Physical Chemistry Letters (2021).
by Aditi Singh
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