Wohlgemuth Method

WOHLGEMUTH METHOD (Wohlgemuth) for the determination of amylase is based on establishing the limiting amount of the substance under investigation (saliva, blood, urine, pancreatic juice, etc.) that is still capable, under defined experimental conditions, of completely splitting a known amount of starch down to the stage of achroodextrins, i.e., until the reaction with iodine disappears. Wohlgemuth proposes two modifications: the half-hour and the 24-hour method. In both cases, sequentially decreasing dilutions are prepared from the test material in a series of test tubes such that 1 cubic centimeter of water is placed in all tubes; 1 cubic centimeter of the investigated fluid is added to the first, mixed, 1 cubic centimeter of the mixture is transferred to the next tube, also mixed, and 1 cubic centimeter is transferred to the next, and so on until the last tube. Then, in the 24-hour modification, 5 cubic centimeters of a 1% starch solution and a little toluene are added everywhere, and the tubes are left for a day in a thermostat, after which tap water is added almost to the top of each tube along with 0.1 normal iodine solution drop by drop until a clear coloration is obtained. The tube is noted in which the blue or violet coloration characteristic of starch does not appear, but only the reddish-brown coloration characteristic of dextrins is present. The amylolytic power is expressed by the number of cubic centimeters of 1% starch that can be split under the conditions of the experiment by 1 cubic centimeter of the test material. If, for example, the limit (disappearance of the iodine reaction, i.e., the splitting of 5 cubic centimeters of starch solution) is reached in the 4th tube, where there was 0.0625 cubic centimeters of the test substance, then 1 cubic centimeter of the latter is able to split an amount of starch corresponding to the proportion: 0.0625:5 = 1:x, whence x = 80. This latter value serves as a measure of enzyme activity and is designated as D^37°_24h, where the upper and lower indices indicate the experimental conditions (duration and temperature). In the half-hour procedure, instead of 5 cubic centimeters of 1% starch, 2 cubic centimeters of 0.1% starch are taken, and the tubes are placed not in a thermostat, but in a water bath at 38° for more uniform heating. The calculation is carried out in a completely analogous manner; the amylolytic power is expressed in cubic centimeters of 0.1% starch and is designated by the symbol d^38°_30m. The Wohlgemuth method, which has become widespread, must be recognized as suitable only for very rough, approximate determinations, because it allows for the consideration of only very sharp fluctuations in the quantity or activity of the enzyme (on the order of 50-100%). More accurate results are yielded only by methods that account for amylase action by the amount of sugar formed.