Determination of the Effect of Minor Chemical Modification of β-Galactosidase on Electrophoretic Mobility and Development of a Stereospecific Method for the Analysis of Tartaric and Malic Acids

Abstract

Studies in single molecules are a relatively new field of research in biochemistry. Research in this area looks at how enzyme activity can vary compared to a typical ensemble environment. This field of study covers a wide variety of environments where single molecules can be located, such as in a gaseous mixture in spectroscopy experiments or inside the body. Single molecule enzymology has grown greatly because there has been an interest in technology to measure the activity of single molecules. Ensemble methods of analysis only provide information about the average state of a large number of molecules, whereas in methods such as capillary electrophoresis, the researcher can see the active state of each individual protein molecule. However, dynamic conformation changes in ensembles can mask individual changes in structure or any small changes that do not conform to the average. Suppose one is only looking at the state of a single enzyme. In that case, there is a lot of information that can be gained from that single enzyme such as conformation and activity; as well as intermediates can easily be detected. An important aspect of these studies to note is that in large scale enzyme studies, there are also interactions between the molecule of interest and the solvent. The other molecules in solution are not typically of interest but they can easily influence the characteristics of the molecule of interest due to chemical bonding. This bonding is important for catalysis studies but is not vital to any studies of single enzyme molecules, as the substrate bonding data is not being collected. Related to this, during the reaction in a large-scale enzyme setting, there are instances where multiple turnovers may occur, and also, not all the enzyme molecules will be in the same active state during these reactions. This is not a concern in single molecule reactions as the reaction sequence of each enzyme molecule can easily be observed. This is particularly important in reactions that involve protein folding and DNA synthesis. Studies have utilized the enzymes β-galactosidase and alkaline phosphatase, and have shown that many different types of information can be gathered from the study of an individual molecule. For example, a study has looked at how temperature affects the active conformations of these enzymes. This study showed that an enzyme has many different active conformations that can be observed, and that heating the enzyme can induce a conformational change. However, as is already known, an enzyme can only be heated to a certain point, as there is a process of denaturation.