Charles A. Zittle

Binding of calcium to casein: Influence of pH and calcium and phosphate concentrations

Abstract The binding of calcium to casein has been determined, both by analysis of the aggregated casein and by equilibrium dialysis. Values for maximum amount of calcium bound and dissociation pK values are reported for pH 7.0 and compared with values in the literature. Calcium is not bound to casein at pH 5, but binding does occur and increases as the pH is raised. The increase in binding parallels the increase in net negative charge of the casein, as well as the degree of ionization of a substituted phosphoric acid up to about pH 7. The time that the calcium caseinate aggregates are permitted to form does not affect the calcium binding. Aggregates formed at higher temperature in part redissolve at lower temperatures, but the binding of calcium per unit weight of aggregate is unchanged. In casein systems containing both calcium and phosphate, binding of the latter occurs only above pH 6, and simultaneously the binding of calcium increases greatly. The isoelectric point (maximum precipitation) of casein is not changed in the presence of calcium chloride.

Purification and properties of acid phosphatase in bovine milk

Abstract The acid phosphatase of bovine milk has been purified over 40,000-fold by extensive use of Amberlite IRC-50. The enzyme is a basic protein with an optimum pH of 4.75. Ascorbic acid triples the activity, while other reducing agents have only a slight activating effect. The acid phosphatase acts on aromatic phosphates, casein, and pyrophosphates (both organic and inorganic). It did not act on AMP, serine phosphate, and glycerol phosphate.

Ribonuclease in bovine milk

Abstract The ribonuclease of milk has not been investigated, although there has been in recent years a rapid expansion of our knowledge of ribonuclease in body fluids such as cerebral spinal fluid ( Houck, 1958 ), blood ( Levy and Rottino, 1960 ; Rabinovitch and Dohi, 1957 ; Zittle and Reading, 1945 ), urine ( Levy and Rottino, 1960 ), and the extracellular fluid of skin ( Tabachnick and Freed, 1961 ). Zittle and DellaMonica (1952) noted that certain purified fractions of bovine milk showed phosphodiesterase activity when ribonucleic acid was used as the substrate. Bailie and Morton (1958) showed that the nucleic acid content of mammary gland microsomes diminished when the microsomes were incubated in milk serum for 12 hours, and suggested that the phosphodiesterase of Zittle and DellaMonica (1952) might have caused the decrease in nucleic acid. They suggested that milk microsomes (with a low nucleic acid content) might be derived from mammary gland microsomes, which contained a much higher nucleic acid content. In this report evidence is presented for the presence of relatively high concentrations of ribonuclease in cows milk. Some properties of this enzyme, as well as its partial purification, are described.

Purification of human red cell acetylcholinesterase

Abstract The acetylcholinesterase in human red blood cells was extracted from the stromata by means of the surface-active substance polyoxyethylene sorbitan monolaurate (Tween 20). After a series of purification steps, preparations dried in the frozen state were freed of Tween by extraction with acetone and freed of lipide by extraction with n-butanol or ethanol. The dry powders, representing a 250-fold purification, were stable at 7 °. Electrophoresis on paper showed that the isoelectric point of the esterase lies below pH 6.0.

Solubility transformation of α-lactalbumin

Abstract The ability of α-lactalbumin to occur in two forms differing in solubility appears to depend on the binding of anions. In dialyzed solutions α-lactalbumin exists preponderantly (75%) in a form insoluble in 2 M ammonium sulfate. In the presence of 0.1 M NaCl or a variety of other salts it is transformed largely (89%) to a form soluble in 2 M ammonium sulfate. These two forms do not differ in the ultracentrifuge; in electrophoresis at 0.1 ionic strength α-lactalbumin is homogeneous. A shift of the isoelectric point, determined by precipitation and titration, from pH 4.8 in salt-free solutions to pH 3.6 in 0.5 M NaCl suggests that the chloride anion, and presumably other anions, are strongly bound by α-lactalbumin. The binding of anions suggests that the solubility transformation may be due to a slight change in physical configuration implemented by the anion binding. The study of precipitation in the isoelectric region has shown also that 0.1 M NaCl salts-in the α-lactalbumin to some extent (about 1 mg./ml.); this effect is no longer apparent at 0.5 M NaCl concentration.

Separation of proteins by gradient solvent extraction of a protein precipitate

Abstract A mixture of bovine serum albumin and human hemoglobin is used to show the applicability of the gradient extraction method for the fractionation of proteins. The mixture is precipitated with ammonium sulfate and serially extracted with ammonium sulfate solution continuously diluted with water. The method has also revealed a reduction in the solubility of α-lactalbumin that had been dried from the frozen state and stored at 25 °. Equipment for accomplishing the continuous dilution is described as well as calculations of the concentrations to be expected.

Action of rennin and pepsin on α-casein: Paracasein and soluble products

Abstract α-Paracasein, resulting from the action of rennin and pepsin on α-casein, and separated by precipitation at pH 4.7, is a heterogeneous substance. Fractionation and paper electrophoresis show it to contain a major component and several minor components, and Tiselius electrophoresis shows that even the major component is complex when the enzymes have acted for 3 hours. The soluble peptide fraction is very heterogeneous and contains 10–12 or more components. Extraction of both paracasein and peptide fractions with the 4:1:1 solvent mixture ( n -butanol-acetic acid-water 4:1:1) is a useful procedure for separating fractions (soluble and insoluble) differing in composition. The glucopeptide described by Nitschmann et al . (8) was separated from other components by this means. The enzymes rennin and pepsin release the same major products, but there are differences in the minor products formed. The latter suggests that pepsin has the broader specificity.

Reactivity of the alkaline phosphatases of bovine milk and intestinal mucosa with the substrates phenyl phosphate and o-carboxyphenyl phosphate.

Abstract Bovine intestinal mucosa alkaline phosphatase reacts equally with the substrates phenyl phosphate and o -carboxyphenyl phosphate. Milk alkaline phosphatase, on the other hand, at a concentration of the substrates of about 8 × 10 −5 moles/l., is 1 3 to 1 4 reactive with the o -carboxyphenyl phosphate as with phenyl phosphate. Analyses of these reactions with a range of substrate concentrations showed that the difference in reactivity is due to a difference in enzyme-substrate dissociation constants, K m . The p K m values for the mucosa phosphatase with both substrates at pH 9.7 is 3.7; with the milk phosphatase and phenyl phosphate the p K m value is 4.6, whereas with o -carboxyphenyl phosphate the value is 2.8. The K m values were also determined for a range of pH values. The difference noted above persisted throughout. K m values obtained with the milk phosphatase in a lipide-containing complex were identical with values obtained with the phosphatase after dissociating with n -butyl alcohol.

Purification of human red cell acetylcholinesterase by electrophoresis, ultracentrifugation and gradient extraction

Abstract Purification of human red cell cholinesterase by means of electrophoresis on paper, ultracentrifugation, and gradient exctraction is described. By these means a protein is isolated that appears to be homogeneous in gradient extraction and free electrophoresis; however, reasons are given for believing that this is not the cholinesterase protein but a major protein with which the cholinesterase is tenaciously associated.

The stabilizing fraction of α-casein

Abstract The quantitative relation of soluble N and P released from α-casein, treated with the enzyme rennin, has been studied. The amounts of soluble N and P are correlated with the calcium-sensitivity of the α-casein. The calcium-insensitive α-casein contains a larger amount of the protective fraction than does the calcium-sensitive α-casein. These findings support the Linderstrom-Lang (2, 3) and Holter (4) protective colloid theory of the clotting of casein by rennin.