Forestry Sciences Laboratory, Northeastern Forest Experiment Station, Forest Service, United States Department of Agriculture, Box 640, Durham, New Hampshire 03824.
Plant Physiol. 1977 Nov;60(5):787-93. doi: 10.1104/pp.60.5.787.
Several improved techniques for isoelectric focusing of isoenzymes in polyacrylamide gel slabs were developed. Using these techniques, three commercial sources of horseradish peroxidase were each examined with three commercial sources of carrier ampholytes to determine their respective isoenzyme profiles.A much higher degree of isoenzyme resolution was obtained than reported previously. A composite of all of the various tests gave a total of 42 peroxidase isoenzymes. Commercial sources of horseradish peroxidase showed many similarities in their isoenzyme patterns, but their differences were sufficient to recognize each source easily. Isoenzyme patterns for all three sources spanned the entire pH range with all three sources of wide range carrier ampholytes. Only a few isoenzymes stained darkly. Most isoenzymes stained moderately to lightly, but all were well resolved. Gels stained for indoleacetic acid oxidase activity showed the same pattern as gels stained for peroxidase activity. This was true for all three commerial sources of the enzyme. The isoenzyme patterns obtained in each run were entirely reproducible, and linear pH gradients were obtained in all cases. Limitations in the pH range of the wide range carrier ampholytes relative to the isoelectric points of the extreme anodic and cathodic isoenzymes led to the adoption of a modified definition of focusing time. In addition, the labeling of isoenzymes commonly used in electrophoresis was adapted for labeling isoenzymes resolved by isoelectric focusing.
几种改进的同工酶聚丙烯酰胺凝胶板等电聚焦技术被开发出来。使用这些技术，三种不同来源的辣根过氧化物酶分别与三种不同来源的载体两性电解质进行了检测，以确定它们各自的同工酶图谱。与之前报道的相比，获得了更高程度的同工酶分辨率。所有不同测试的组合共得到了 42 种过氧化物酶同工酶。辣根过氧化物酶的商业来源在同工酶图谱上有许多相似之处，但它们的差异足以轻易识别每个来源。所有三种来源的同工酶图谱都涵盖了整个 pH 范围，所有三种来源的宽范围载体两性电解质都是如此。只有少数同工酶染色很深。大多数同工酶染色中等至浅色，但都有很好的分辨率。用于吲哚乙酸氧化酶活性染色的凝胶与用于过氧化物酶活性染色的凝胶显示出相同的模式。这对于该酶的所有三种商业来源都是如此。每次运行获得的同工酶图谱完全可重复，并且在所有情况下都获得了线性 pH 梯度。宽范围载体两性电解质的 pH 范围相对于极端阳极和阴极同工酶的等电点的限制导致采用了聚焦时间的改进定义。此外，用于电泳的同工酶的标记被适应于通过等电聚焦解析的同工酶的标记。
