The effect of temperature on the rate of Kjeldahl digestions in the absence of catalyst and oxidizing agent has been studied. Both the clearing time and the minimum time for complete recovery of nitrogen are markedly decreased by raising the digestion temperature. The appreciable rise in temperature during prolonged digestions and the effect of time and temperature on the pyrolytic loss of nitrogen are considered. By proper choice of digestion conditions nitrogen can be completely recovered in a reasonable time even from refractory compounds. The time may be further decreased by the use of mercury as catalyst. The use of hydrogen peroxide as an oxidant in Kjeldahl digestions is discussed and the effects of the volume and number of additions at various temperatures after different cooling times determined. Earlier claims regarding complete recoveries with few additions cannot be substantiated. A modified micro-apparatus for the distillation of ammonia from Kjeldahl digestions is described and acidimetric methods for the determination of the ammonia are critically examined. As a result of this work it is possible to develop procedures for the Kjeldahl determination of nitrogen in various materials. A rapid and precise method for the determination of 0.2-2 mg of nitrogen in amino acids and proteins is described.
Sedimentation-equilibrium studies are made of the moleoular weights of bovine ,B.laotoglobulins A, B, and C at pH 7·5. The order of dissooiation of dimer to monomer is A> B ~ C. The dissooiation oonstant (Kd) for A and B is 0·6 X 10-4 and 0·08x 10-4 mole 1-1 at 20°C, respeotively. The oonditions ohosen for these measurements are based on sedimentation velooity studies in the pH range 6-9. There is no ohange in sedimentation velooity behaviour following different times of standing at 20°C for pH < 7·5. The sedimentation patterns exhibit a single peak with some trailing on the solvent side. At low oonoentrations the plot of weight average sedimentation coefficient (,~) versus concentration (0) is in aocord with that of a rapidly dissociating system of monomer-dimer type. There are time-dependent aggregations above pH 8. Effects of changes in ionic strength and addition of methanol are considered.Using several values of K d , .9 versus 0 ourves are oaloulated by the method of Gilbert (1963) and oompared with the present experimental ourves and also those of Zimmerman, Barlow, and Klotz (1970). The agreement between theory and experiment is only moderate.
The products of reaction of bovine p-lactogiobulin variants in urea at pH 3-9 were examined in two types of starch gel electrophoresis system. The first type, containing 7M urea at pH 3·5, is designed to cause minimum reversal and minimum aggregation of the products during the electrophoresis. The second type, without urea present, helps differentiate between reversible and irreversible products. The formation of irreversible products, giving rise to slower moving bands in electrophoresis, increases with increasing reaction time, urea concentration, and pH. These products are not formed when sulphydryl reagents are present. They arise from intraand intermolecular -SH/-SS-interchange, even at pH 3 ·0. After removal of urea the products were fractionated by chromatography on Sephadex G100. In the early stages of reaction a large proportion of product may be reversed to a form indistinguishable from the native protein in molecular size, mobility, and optical rotatory dispersion. Later, production of dimer (mol. wt. 36,000), trimer, tetramer, etc., in which there has been -SH/-SS-interchange, and which cannot revert completely to the native conformation, occurs. Denaturation is considered to proceed via the monomer unit.
The heterogeneity of casein is discussed in the light of methods currently used for the fractionation of casein. In particular, the possible heterogeneity of certain preparations of α-casein is considered. This is important because it has been generally considered that α-casein is the protective colloid which is altered when the enzyme, rennin, acts on casein micelles. Recently, Waugh and von Hippel (1956) have suggested that their new component x-casein, and not α-casein, is the protective colloid. These two viewpoints could be reconciled if α-casein samples previously examined contained x-casein as well. In the present work, a study is made of filter paper electrophoresis, micelle-forming properties, and sedimentation of casein fractions. It is shown that x-casein is concentrated with α-casein in fraction A during the alcohol fractionation method of Hipp et al. (1952). On the other hand fraction B contains α-casein essentially free of x-casein. The a-casein obtained in the urea fractionation method of Hipp et al. also contains x-casein. Thus only alcohol fraction B is a suitable source of pure α-casein. During the paper electrophoretic examination of casein fractions a number of minor protein components are observed. A component moving more slowly than γ-casein is present in acid casein, second-cycle casein-fraction P, and an alcohol fraction. This component was first observed in the latter fraction by Hipp et al. (1952) when preparing γ-casein. Electropherograms of second-cycle casein-fraction S indicate the presence of x-, β-, and γ-casein, and two minor components moving between x- and β The way in which these components arise is briefly discussed.
α-, β-, and x-casein are aggregated at neutral pH and room temperature in salt solution. They may be disaggregated at high pH or at neutral pH in concentrated urea solution. At neutral pH in salt solution, α-casein forms aggregates centring around a preferred size. As the pH is increased the size decreases until at pH 11 (I 0.20) it is disaggregated completely. Measurements of the molecular weight by sedimentation and diffusion at pH 11 give a value of 24,800�1000. Approach to sedimentation-equilibrium measurements at pH 12, using the Archibald method, gives a value of 25,500�1000. In 6M urea solution at pH 7.3 sedimentation and diffusion give a value of 27,600�1000. At room temperature and neutral pH, β-casein is present as single molecules in equilibrium with an aggregate of very high molecular weight. At pH 11, it is disaggregated with a molecular weight of 17,300�800 (by sedimentation-diffusion). At pH 7 in 631 urea a value of 19,800�1000 is obtained for the molecular weight. Preliminary measurements by the Archibald method at pH 12 give a value of 26,000�3000 for the molecular weight of x-casein. These results are discussed in relation to those of other workers.
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