THE PURPLE COLOR REACTION GIVEN BY DIPHENYLAMINE REAGENT .1. WITH NORMAL AND RHEUMATIC FEVER SERA
THE PURPLE COLOR REACTION GIVEN BY DIPHENYLAMINE REAGENT .1. WITH NORMAL AND RHEUMATIC FEVER SERA
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DOI：
10.1172/jci102492
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发表时间：
1951-01-01
影响因子：
15.9
通讯作者：
HESS, EL
中科院分区：
文献类型：
--
作者：
AYALA, W;MOORE, LV;HESS, EL
8试剂组成：冰醋酸100ml.硫酸2.75毫升，二苯胺1克。不适合比色读数。用20%三氯乙酸溶液沉淀血清蛋白时，沉淀得到的酸性水解物中有紫色反应，滤液中没有。产生紫色C4颜色的部分只有在酸或碱水解后才能从血清中透析出来。在Niazi和State(1)描述的程序中，用20%的三氯乙酸溶液从血清中沉淀蛋白质，上清液去掉，沉淀物用5%的三氯乙酸溶液水解，离心后得到的透明上清液用二苯胺试剂检测。由于从血清中提取的20%邻三氯乙酸滤液中不含任何干扰物质，因此直接对血清进行水解法是令人满意的。对水解步骤的研究表明，紫色反应的强度随着时间的延长而增加，直到20分钟，然后缓慢下降，几个小时后变为负值。为此，我们采用20分钟作为标准的水解液时间。加热血清水解液和试剂的时间是影响所产生的颜色强度的另一个重要因素。色强度(以530MJ处的光密度表示)与加热时间的关系图显示，在加热到2小时时，随着时间的延长，色强度迅速增加，之后增加的速度很小。当继续加热时，紫色移到蓝色(两个小时后明显可见)，吸收曲线除了在530mFL处有一个最大吸收带外，还在650 MJU处出现一个新的吸收带。吸收曲线的形状随加热时间的变化如图1所示。我们选择30分钟作为标准加热时间；此时获得强烈的紫色，几乎不受蓝色带的干扰，如图1所示。
8 Composition of the reagent: glacial acetic acid 100 ml., conc. sulfuric acid 2.75 ml., diphenylamine 1 gm. tion unsuitable for colorimetric readings. When serum proteins are precipitated with 20% trichloroacetic acid solution, the purple color reaction is present in the acid hydrolyzate obtained from the precipitate and absent in the filtrate. The moiety giving the purple c4olor is dialyzable from serum only after acid or alkaline hydrolysis. In the procedure described by Niazi and State (1), the proteins are precipitated from serum with a 20% o trichloroacetic acid solution, the supernatant discarded, the precipitate hydrolyzed with a 5% trichloroacetic acid solution, and the clear supernatant obtained after centrifugation is tested with the diphenylamine reagent. Since the 20% o tri-chloroacetic acid filtrate from serum does not contain any interfering substance we have found it satisfactory to hydrolyze the serum directly. A study of the hydrolysis step shows that the in-tensity of the purple color reaction increases with time up to 20 minutes and then decreases slowly and becomes negative after several hours. For this reason we have adopted 20 minutes as a standard time of hydrolysis.The time of heating the serum hydrolyzate and the reagent is another important factor in the in-tensity of the color produced. A plot of color in-tensity (in terms of optical density at 530 mj,) against time of heating shows a rapid increase with time up to two hours, after which the rate of increase is slight. When the heating is continued, the purple color shiftsto blue (clearly noticeableat two hours) and the absorption curve develops a new band with amaximum at 650 mju in addition to the one at 530mFL. The variation in the shape of the absorption curves with time of heating is presented in Figure 1. We have selected 30 min-utes as a standard timeof heating; at thistime an intense purple color is obtained with little interference from the blue band, as can be seen in Figure 1.