The use of the thermodynamic formalism in the investigation of biochemical reactions constitutes one of the key analysis in bioenergetics, and the first step in such analysis is the selection of the adequate reference state. For biochemistry majors, thermodynamic analysis based on the chemical reference state is used in Physical Chemistry courses, while the biological and biochemical reference states are used in Biochemistry courses. As these definitions are introduced in different courses, it is difficult that students can understand the need to select a reference state as a first step in the energy analysis of a system. The lack of suitable examples in textbooks to illustrate the importance of the adequate selection of the reference state in a thermodynamic analysis, promoted the present analysis of the energetic role of pyrophosphate (PPi) in comparison with adenosine-triphosphate in different ambient conditions, namely, the early PPi world (better described by the chemical reference state), the enclosed systems like the cells (better described by the biological reference state), and the actual thioester world (better described by the biochemical reference state). This example not only provides a new interesting point of view on the evolution of two biochemical fuels but also represents a biochemical example in which the use of different reference states can illustrate a single process from different points of views.
Keywords: biochemical evolution; general education for science majors; thermodynamics.
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