Specificity of the Kober Reaction

Abstract

THE specificity of the Kober^1,2 reaction for oestrogen assay has been studied as part of a general investigation of the mechanisms of steroid acid colour reactions. Exactly what constitutes a positive Kober reaction has never been denned; in this work, a Kober positive steroid is regarded as one which produces pink chromogen absorbing maximally in the 500–555 mµ region by the Brown^3,4 or Ittrich⁵–Bradshaw⁶ procedures. This chromogen we have designated χ⁴ because at least three other chromogens absorbing at shorter wavelengths are produced by the Kober reaction with various oestrane derivatives. On this basis, from a study of 116 relevant steroids, the following features of the steroid molecule were found to be simultaneously necessary if the steroid is to give a positive Kober reaction. An oxygen function (carbonyl or hydroxyl) is essential at either C16 or C17 and is permissible at both. The only exceptions are the Δ16-steroid discussed later and 16-oxoestrone; oestroiie-16 is only feebly positive. Of the fifteen possible combinations of oxygen function at C16 and/or C17, thirteen have been tested and all except the two just mentioned are strongly positive and give a pink chromogen with ε_max of 30,000 to 60,000.