Just a few years before Harden and Young’s discovery of NAD, Louis Pasteur had shown that yeast cells were responsible for fermentation, the process in which yeast cells consume sugars and convert them to alcohol and other products. Fermentation is responsible for creating the air pockets in your bread and giving wine and beer both their alcohol content and their unique flavors. Fermentation in yeast is also the same as one of the metabolic processes that occurs in animals and humans to generate energy. In his groundbreaking work, Arthur Harden sought to learn more about how yeast perform fermentation, so they tried to reproduce the process outside of the yeast cells. Using laboratory techniques, they were able to break open yeast cells and separate their contents into two fractions, one that one was sensitive to heat (i.e. heat destroyed its ability to perform the fermentation reaction) and another that wasn’t. By separating and then recombining the fractions, Harden and Young were able to show that the fermenting ability of the heat-labile fraction depended on the heat stable fraction. They surmised that the heat labile fraction contained a protein or proteins responsible for fermentation, and the heat stable fraction contained co-factors (like NAD) and other stable molecules that helped the proteins perform the reactions.   http://rspb.royalsocietypublishing.org/content/78/526/369