Dabsyl-Quenched Fluorescent Probes to Study the Acid-Base Properties of Enzymes: The Fox Grape Effect
The use of fluorescent indicators to measure enzyme activity has been known for decades, however, one major limitation to the measurement of enzyme acid-base properties are the quenching mechanisms used to reduce background fluorescence. Quenching mechanisms, such as Dabsyl, have been found to be the most reliable and effective, allowing the measurement of enzyme acid-base proneities with greater accuracy and sensitivity.
In order to understand the acid-base properties of enzymes, researchers have developed a method termed the Fox Grape Effect, which was first described in a Japanese paper (1). The technique utilizes a quenched fluorescent probe by modification with a quencher to reduce background fluorescence. The probes used in this technique are modified with either a single or a mixture of two quenchers. By combining different quenchers together, researchers are able to create probes that can detect different levels of enzyme activity at different pH ranges.
The Fox Grape Effect is based on the principle that the ratio of the activity of two quenchers (Dabsyl and 4-acetyl-4-hydroxy-4-thiocyclohexyl)propenoic acid (ACHA)), can be used to measure the extent of enzyme activity. When the two quenchers are combined, the probe become more sensitive to the pH of the environment that the enzyme is being actuated in. When the pH decreases, so does the efficiency of the two quenchers and a second quencher is used to help reduce background fluorescence. In order to achieve this, researchers use a dip-dye method to measure the enzyme activity by monitoring the decrease in fluorescence intensity when the pH is increased or lowered.
In order to measure the activity of the quenchers, researchers employ the use of a fluorescence reader (FR) that is calibrated for the brightness of the probe. This FR is used to measure the absolute fluorescence (RF), which is then used to compare the activity of the quenchers. If a difference between the two quenchers is observed (an increase or decrease in fluorescence intensity), then the presence of the enzyme has been demonstrated. The enzyme is then characterized by the difference in fluorescence intensity throughout the pH range that is used.
The Fox Grape Effect is an important tool in enzyme research and has been utilized in a number of studies including the determination of the catalytic properties of a variety of enzymes such as β-galactosidase (2) and cycloheximidease (3). The excellent sensitivity and accuracy of the technique make it a popular method for the characterization of enzymes. Furthermore, the technique can be used to measure the activity of enzymes at different pH conditions, which can be extremely useful in studies looking to characterize the pH-dependent behavior of various enzymes.
The use of fluorescent probes to measure enzyme activity is an effective and very accurate tool. The Fox Grape Effect offers a unique approach for the evaluation of the acid-base properties of enzymes and is an important tool for researchers in understanding the nature of enzyme activity and behavior. The technique is an important and invaluable tool for researchers who are characterizing the behavior of enzymes and the activity of various enzymes, particularly those that are sensitive to their environment.
