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INTRODUCTION. CULBERTSON (1932) has recently described a method for estimating the antibody content of antisera against crystalline egg-albumin. He determined by tests on supernatant fluids the point at which antigen and antibody neutralised each other. At this neutralisation point he found that antigen and antibody combined in a ratio of 1 to 13, and that the amount of precipitate nitrogen was maximal. The ratio 1 to 13, he stated, held for all the precipitates in the post-zone where antibody was in excess.The optimal proportions method of titrating either antigen or antibody in the precipitation reaction, described for horse serum and its antisera by Dean and Webb (1926), was believed to indicate the proportions of the reagents which neutralised each other. In contrast to Culbertson's finding Dean and Webb reported that maximal precipitation occurred not at their optimal or neutral point, but where excess of antigen was present.The present communication records studies made to establish the relationships of the points of neutrality, constant-antibody (Dean and Webb) optimal particulation, and maximal precipitation. The point of maximal precipitation for antisera to crystalline egg-albumin has been determined with precautions calculated to increase the accuracy of the measurements. Additional experiments have been carried out with antisera to crystalline horse-serum albumin. In the experiments of Culbertson and of Dean and Webb the amount of an antiserum was held constant and that of the antigen varied. In order to obtain a more rigorous test of Culbertson's conclusion that there is a constant ratio of antigen nitrogen to precipitate nitrogen of 1 to 13 in the zone of

1. The amount of crystallisable albumin in egg-white and the amount of total globulin in horse serum have been estimated by means of the precipitation reaction. The results are in good agreement with those obtained by other methods.2. For such estimations it appears advisable to use only antisera prepared against individual proteins.3. A description of the manufacture and the characteristics of the pure proteins used is appended.

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The apparent densities of proteins in aqueous solutions have been studied by a number of investigators, including Chick and Martin (1913), and, more recently, by Svedberg and his colleagues, but comparatively few observations of the densities of protein crystals have been published. Eyer (1932) gave the value 1·315 for insulin, Crowfoot (1935) 1·306; Adair and Adair (1934) found 1·296 for haemoglobin; Bernal and Crowfoot (1934) gave 1·28 for pepsin. The media used for the measurements on pepsin and insulin were not recorded. A method for measuring the densities of small crystals by means of observing their flotation in media of different densities was described by Retgers (1889). The present communication records a modification of his method adapted for the investigation of the density of crystalline and of denatured proteins. The first problem studied was the choice of suitable media. The mixtures of organic liquids which are generally used for density determinations of inorganic crystals are open to objections on the grounds that protein crystals cannot be freed from films of their mother liquor without grave risks of irreversible changes. In working with aqueous media this difficulty need not arise, but the media chosen must fulfil the somewhat exacting requirements of a high density with freedom from the capacity to denature or to dissolve the protein.