Enzymatic Methods of Analysis

the methods for the quantitative determination of chemical substances in solution based on the use of enzymes. Enzymatic methods are employed to determine the quantity of substances that are capable of taking part in chemical reactions catalyzed by enzymes, as well as of substances that are activators or inhibitors of enzymes. The methods are characterized by high sensitivity and specificity since enzymes catalyze the conversion of substances with great speed and selectivity. These characteristics hold even when the compound to be analyzed is mixed with other substances similar in chemical structure.

In making a determination of the substrate in an enzyme-catalyzed reaction, an enzyme and the other components necessary for the réaction are added to the sample. When the reaction is completed, the quantity of the reaction product in the solution is measured. For example, the determination of ethanol in a solution using the enzyme alcohol dehydrogenase is carried out in the presence of the enzyme’s coenzyme, nicotinamide adenine dinucleotide (NAD). During the enzyme-catalyzed reaction,.the latter is converted quantitatively into reduced NAD, which, unlike the oxidized form, is able to absorb ultraviolet light at a wavelength of 340 nanometers. By measuring the absorption, it is possible to establish the concentration of the reduced NAD and calculate the concentration of ethanol. This method permits determinations of 1 microgram (xg) of alcohol in 1 milliliter of solution.

Many enzymatic methods of analysis are based on changes in the acidity of the solution during an enzyme-catalyzed reaction. For example, the esters of carboxylic, phosphoric, and other acids can be determined using the specific enzymes that catalyze their hydrolysis. Since hydrolysis is accompanied by the formation of corresponding acids, titration of the acids at the end of the reaction permits a calculation of the concentration of the ester that is being determined.

Enzymatic methods of analysis often involve combinations of enzyme-catalyzed reactions (coupled reactions). For example, the concentration of glucose can be determined using the enzymes glucose aerodehydrogenase and peroxidase. Under the action of the former, glucose is converted into gluconic acid; in addition, hydrogen peroxide is formed, which, under the subsequent action of peroxidase, can oxidize the o-dianisidine (or tolidine) added to the solution and impart a color. By measuring the intensity of the color, it is possible to calculate the initial glucose concentration (sensitivity, 5 µg). This method is used for the rapid determination of glucose in the urine of diabetics by means of test paper impregnated with the necessary reactants.

Kinetic methods of analysis, which form a separate group, are based on the relationship between the rate of an enzyme-catalyzed reaction and the concentration of the substance being analyzed, which with kinetic methods may be a substrate, an activator, or an inhibitor. If the nature of this relationship is known, the concentration of the substance being analyzed can be calculated by measuring the rate of the reaction. For example, the quantitative determination of organophosphorus insecticides, which are strong inhibitors of the enzyme cholinesterase, is carried out by measuring the activity of the enzyme in the absence and in the presence of the inhibitor. The sensitivity of this method is sufficient to determine 0.015 µg of diethyl para-nitrophenyl phosphate in a sample; for magnesium ions (based on their activating effect on the enzyme oxidizing isocitric acid), the sensitivity is 0.1 µg.

Enzymatic methods of analysis using enzymes firmly attached to such solid carriers as polymers, inorganic sorbents, and gels have become common. Here, immobilized enzymes are placed on electrochemical transducers (glass, platinum, and other types of electrodes); these enzyme electrodes serve as instruments for measuring the rate of an enzyme-catalyzed reaction in a solution of the substance under analysis. Enzyme electrodes are used to determine urea, amino acids, penicillin, and glucose; they are sensitive to 0.1–0.01 µg in a sample.