Theerasilp S, Kurihara Y
Department of Chemistry, Faculty of Education, Yokohama National University, Japan.
J Biol Chem. 1988 Aug 15;263(23):11536-9.
The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. Previous attempts to isolate miraculin from deeply colored alkaline extracts of the miracle fruit were unsuccessful. We found that miraculin is extracted with 0.5 M NaCl solution. The extracted solution is colorless and shows the strong sweet-inducing activity. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose affinity chromatography. The purified miraculin thus obtained gave a single sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure. The sample also gave a single band having molecular weight 28,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was much lower than the values reported previously (40,000-48,000). The amino acid composition of the purified miraculin was determined. Sequence analysis of the purified miraculin indicated that it is composed of a pure single polypeptide and identified 20 amino-terminal amino acids. The purified miraculin contained as much as 13.9% of sugars, which consisted of glucosamine, mannose, galactose, xylose, and fucose in a molar ratio of 3.03:3.00:0.69:0.96:2.12.
味觉修饰蛋白奇甜蛋白具有将酸味转变为甜味的独特特性。此前从神秘果的深色碱性提取物中分离奇甜蛋白的尝试均未成功。我们发现奇甜蛋白可用0.5M氯化钠溶液提取。提取的溶液无色，且具有很强的诱导甜味的活性。通过硫酸铵分级分离、CM - 葡聚糖凝胶离子交换色谱和伴刀豆球蛋白A - 葡聚糖凝胶亲和色谱从提取液中纯化出奇甜蛋白。如此获得的纯化奇甜蛋白在反相高效液相色谱中给出单一尖锐峰，表明其纯度很高。该样品在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中也给出一条分子量为28,000的单一谱带。这个值远低于先前报道的值（40,000 - 48,000）。测定了纯化奇甜蛋白的氨基酸组成。纯化奇甜蛋白的序列分析表明它由单一纯多肽组成，并鉴定出20个氨基末端氨基酸。纯化的奇甜蛋白含有高达13.9%的糖类，这些糖类由氨基葡萄糖、甘露糖、半乳糖、木糖和岩藻糖组成，摩尔比为3.03:3.00:0.69:0.96:2.12。
