The hydrogen ion dissociation curve of the crystalline albumin of the hen's egg

Abstract

Tmis paper is a report upon a somewhat extensive investigation of the hydrogen electrode titration of crystalline egg albumin. The work was undertaken with the object of examining the limitations of the method as a means of establishing a quantitative definition of the amphoteric properties of a protein. Crystalline egg albumin was chosen because it is probably the best accredited example of a molecularly homogeneous protein. The extensive work of S0rensen and of his colleagues, of Svedberg and of others indicates that of the common protein preparations egg albumin exhibits the greatest degree of constancy in composition, molecular weight and solubility under varying conditions of preparation and treatment. This protein has the further advantage that it is soluble in water over the whole titratable range of PH.The data which will be considered derive from observations gathered over a period of five years upon five distinct preparations of the protein. These preparations had been submitted to considerable variations in treatment prior to titration. Each was crystallised four times-one by the original method of S0rensen [1917], the others by a modification of this method in which sodium sulphate replaced ammonium sulphate as the salting-out agent. Two of the latter preparations were employed in the electrometric work without having been reduced to the dry state. Two were converted into a dry powder and stored for some time before use. One of the dry preparations was dissolved in water and titrated without further purification, the other after one more recrystallisation. Some details of the method of preparing crystalline egg albumin in the dry state with the aid of sodium sulphate will be found in the experimental section.Each product was prepared for titration as a stock solution which had been dialysed in distilled water until the sulphate ion could not be detected in a dialysate after sixteen hours' contact with the solution. Varying amounts of dilute HCI or of NaOH were added to equal volumes of the stock solution and the mixtures were then diluted to a predetermined concentration of protein. The majority of the observations relate to systems containing 22-25 g. of protein in 1000 g. of water. In a few cases this value was reduced to 15 g. and in a few others raised to 45 g. The PE of each mixture was determined in a rocking hydrogen electrode [Clark, 1928] at 250, using a saturated calomel half-cell and a saturated potassium chloride liquid junction. The calomel half-cell was calibrated with the aid of 0.1 N HCI, for which we assumed a pH of 1-075 [Scatchard, 1925]. The interval between the preparation of a mixture and the determination of its PH did not exceed 1 hour.Commonwealth Fund Fellow. ( 227 )

On the Nature of the Chemical Mediators Involved in Anaphylactic Reactions in Mice

“…Antigen. Three-times recrystallized albumen from hens' eggs, prepared according to the method of Kekwick & Cannan (1936), was used.…”

Relative affinities of the anions of strong acids for wool protein

“…Methods of calculating this quantity for dissolved proteins are all based on differences in pH between solutions of the acid alone and of the acid plus protein. They have been described by Loeb [25], Cohn, Green and Blanchard [8], Kekwik and Cannan [22], and Hitchcock [20], among others, and are summarized in reviews by Hitchcock [21] and by Schmidt [30]. In the present investigation pH measurements were made of serial dilutions of each acid used in the absence of protein as well as in the presence of 0.113 percent by weight of dissolved egg albumin.…”

“…The results of these measurements at 22° C are summarized in table 6 and shown in figure 7. The position of the broken line at the top of this figure, inserted for reference, shows the maximum acid-binding capacity, indicated by the work of Kekwick and Cannan with hydrochloric acid [22]. The crosses distinguish measurements in which incipient precipitation was indicated by a faint opalescence.…”

“…With picric and flavianic acids, next highest in order of affinity, the full correction has been applied; a slight overcorrection may result, especially in the range of pH in which only small amounts of acid are bound. Crystalline egg albumin was prepared by the method of Kekwick and Cannan [22], recrystallized five times, and dialyzed free of sulfate. The resulting stock solution contained 7.02 mg of nitrogen per milliliter and gave a dry residue, on evaporation in a vacuum oven at 100° C, of 45.2 mg/ml.…”

Affinity Entrapment of Oligosaccharides and Glycopeptides Using Free Lectin Solution

“…Ovalbumin was prepared according to a procedure described by Kekwick and Cannan. 16 L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin from bovine pancreas (EC 3.4.21.4), bovine pancreas ribonuclease B, human transferrin, and iodoacetamide were obtained from Sigma-Aldrich, Japan K.K. (Tokyo, Japan).…”