Spencer P W, Spencer R D
Department of Horticulture and Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Plant Physiol. 1974 Dec;54(6):925-30. doi: 10.1104/pp.54.6.925.
The proteolytic activities of alpha-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds (Cucurbita moschata) were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates. Chymotrypsin, trypsin, pepsin, and bromelain exhibited activity against all or almost all of the protein substrates. The activity of 1 mug of alpha-chymotrypsin or trypsin and 100 ng of pepsin could be easily detected by this method of assay within 4 to 5 minutes depending upon the substrate. The enzyme extracted from 3-day germinated pumpkin seeds exhibited strong activity only against pumpkin seed globulin, weak activity against the globulins of squash and cucumber and casein, and no activity against the other protein substrates. Activity against pumpkin globulin was maximal at pH 7.4. When assayed by an increase in ninhydrin-positive products, the enzyme extract from pumpkin seeds also showed strong activity against pumpkin globulin and weak activity against casein. The 1-anilino-8-naphthalenesulfonate-fluorescence method was at least 20 times more sensitive than the ninhydrin method and was 10 to 20 times more rapid.
通过测定α-糜蛋白酶、胰蛋白酶、胃蛋白酶、菠萝蛋白酶以及发芽南瓜种子（南瓜）提取物作用于完整蛋白质底物内部疏水位点结合的1-苯胺基-8-萘磺酸盐释放的能力，来确定它们的蛋白水解活性。酪蛋白、甘油醛-3-磷酸脱氢酶、脲酶、过氧化氢酶、南瓜种子球蛋白和牛血清白蛋白能充分增强1-苯胺基-8-萘磺酸盐的荧光，可用作蛋白水解底物。糜蛋白酶、胰蛋白酶、胃蛋白酶和菠萝蛋白酶对所有或几乎所有蛋白质底物均表现出活性。根据底物不同，用这种测定方法在4至5分钟内可轻松检测到1微克α-糜蛋白酶或胰蛋白酶以及100纳克胃蛋白酶的活性。从3天发芽的南瓜种子中提取的酶仅对南瓜种子球蛋白表现出强活性，对南瓜和黄瓜球蛋白以及酪蛋白表现出弱活性，对其他蛋白质底物无活性。对南瓜球蛋白的活性在pH 7.4时最大。当通过茚三酮阳性产物的增加来测定时，南瓜种子的酶提取物对南瓜球蛋白也表现出强活性，对酪蛋白表现出弱活性。1-苯胺基-8-萘磺酸盐荧光法的灵敏度至少比茚三酮法高20倍，速度快10至20倍。
