ENZYMATIC DEPHOSPHORYLATION OF OVALBUMIN AND PLAKALBUMIN

It has been shown by the work presented in this paper that it is possible to carry out a stepwise dephosphorylation of ovalbumin. With the aid of a prostate phosphatase that attacks only one of the two phosphorus-containing groups in the major component, A1, of ovalbumin, a protein, A2, containing 1 atom of phosphorus per mole has been prepared. Further dephosphorylation with an enzyme of intestinal origin gives a phosphorus-free ovalbumin, A3. Plakalbumin has been similarly dephosphorylated to give P2 and P3. Significant changes in the electrophoretic mobility accompany each dephosphorylation step. This, together with the phosphorus content of the proteins and the crystal form, has been used to characterize and study the five modifications of ovalbumin thus produced

ENZYMIC DEPHOSPHORYLATION OF PEPSIN AND PEPSINOGEN

It has been shown by the work presented in this paper that it is possible to dephosphorylate enzymically pepsin and pepsinogen with a variety of phosphatases. With the aid of a phosphodiesterase and the prostate phosphatase it has been established that the phosphorus in the two proteins is present as a diester and connects two sites of the peptide chain in a cyclic configuration. Removal of the phosphorus does not affect the proteolytic activity against hemoglobin or the synthetic substrate acetyl-L-phenylalanyl diiodotryosine, nor the pepsinogen pepsin transformation. However, an increase of the autodigestion of pepsin is observed

PREPARATION AND PROPERTIES OF A PROTEIN (R ANTIGEN) OCCURRING IN STREPTOCOCCI OF GROUP A, TYPE 28 AND IN CERTAIN STREPTOCOCCI OF OTHER SEROLOGICAL GROUPS

A group A, type 28 protein antigen, resistant to tryptic digestion and previously considered to be a type-specific substance, was purified and its chemical and immunological properties studied. This protein lacks the characteristic properties of a type-specific M antigen since it is apparently unrelated to virulence and does not induce the formation of protective antibodies although precipitins are readily produced. It is designated the R antigen. The R antigen in addition to occurring in "type 28" group A strains also occurs in some strains of streptococci of other serological groups. Protective antibodies, distinct from precipitins for the R antigen, are present in "type 28" antibacterial sera. The antigens responsible for protection have not been identified, and it is possible that several different types may be included among strains designated as type 28 on the basis of the R antigen. The purified R antigen is phosphorus-free and has a sulphur content of 1.04 per cent. In the ultraviolet a maximum absorption was obtained at a wave length of 280 mµ and a minimum at 254 mµ. Electrophoretically the R antigen was found to be 95 per cent homogeneous at most pH values, but at pH 4.6 the main peak separated into two peaks of approximately equal areas, both containing serologically active material. The interpretation of this finding is at present uncertain. The isoelectric pH was at 4.5 in sodium acetate buffer of 0.1 ionic strength. The purified R antigen sedimented in the ultracentrifuge as a single boundary

PREPARATION AND PROPERTIES OF TYPE-SPECIFIC M ANTIGEN ISOLATED FROM A GROUP A, TYPE 1 HEMOLYTIC STREPTOCOCCUS

Type-specific M antigen was extracted by heating type 1 group A streptococci at pH 2 in a boiling water bath. The protein was then purified by digestion with a preparation of crystalline ribonuclease which was free of proteolytic activity. It was further purified by fractional precipitation with (NH4)2SO4. Elementary chemical analysis of the preparation thus obtained showed an absence of phosphorus and a sulfur content of 2.46 per cent. In the ultraviolet the maximum absorption was at a wave length of 276 mµ and the minimum at 255 mµ. In electrophoresis experiments the preparation showed a single peak in the pH range of 3 to 9, but considerable boundary spreading was observed. The type 1 M antigen was isoelectric at pH 5.3 in sodium acetate buffer of ionic strength 0.1. The serological reactivity of the protein isolated was typical of type 1 M antigen. This protein induced the formation in rabbits of type-specific precipitins and protective antibodies. The absorption of type 1 antibacterial serum with the purified M antigen removed both the protective antibodies and the type-specific precipitins from the serum

THE AMINO ACID COMPOSITION OF CRYSTALLINE PEPSIN

The amino acid composition of twice recrystallized pepsin (Worthington Biochemical Corporation) has been determined chromatographically on columns of Amberlite IR 120 resin. The results of the analyses obtained on four different preparations indicate a close agreement in their amino acid composition. Pepsin is unique in that it has a great predominance of acidic amino acids over basic ones. Moreover, all the preparations contain a small and constant amount of hydroxyproline, corresponding to about 0.1 residue per molecule

THE EFFECT OF LITHIUM PERIODATE ON CRYSTALLINE BOVINE SERUM ALBUMIN

A study of the chemical, physicochemical, and immunological changes in bovine serum albumin, brought about by oxidation with lithium periodate, has been made. It has been shown that destruction of certain amino acids occurs, that a change in the absorption spectrum takes place, and that the electrophoretic behavior of the protein is altered. Prolonged contact of bovine albumin with lithium periodate destroys its ability to incite antibodies in experimental animals

AN ELECTROPHORETIC EXAMINATION OF A URINARY MUCOPROTEIN WHICH REACTS WITH VARIOUS VIRUSES

A mucoprotein isolated from human urine and possessing the capacity to react with a number of viruses is electrophoretically homogeneous at pH 6.8 and 8.6. After treatment with influenza virus and elimination of its biological activity, the substance remains homogeneous and its electrophoretic mobility is decreased by approximately 20 per cent

ENZYMATIC DEPHOSPHORYLATION OF OVALBUMIN AND PLAKALBUMIN

In 1927, SCrensen and his collaborators showed that the phosphorus content of crystalline ovalbumin varies somewhat with the preparation and that this protein can be separated by "electrodialysis " into a phosphorus-poor and a phosphorus-rich fraction (1). Direct evidence of the irdaomogeneity of this material was later reported by Longsworth (2) who found two electrophoretic components, Aa and As, in the pH range of 5 to 10. In view of these facts LinderstrCm-Lang and Ottesen (3) suggested in 1949, that if the molecular weight of ovalbumin is taken as 44,000, the phosphorus content does not correspond to an integral number of phosphorus atoms per mole of protein and that this lack of stoichiometry might be explained by the assumption that A1 contains 2 atoms of phosphorus per mole and As 1 atom. The work presented in this report was designed primarily to test this hypothesis by enzymatic dephosphorylation of ovalbumln and to compare the phosphorus content of the resulting proteins with their electrophoretic behavior. As will be shown, these experiments confirm the suggestion of LinderstrCm-Lan

Preparation and properties of typespecific M antigen isolated from group A, type 1 hemolytic streptococcus

Studies of the type-speclfic M antigens of group A streptococci have been continued. In this paper methods for the purification of the M protein of a type 1 strain are described and the chemical and immunological properties of the purified antigen are reported. Materials and Methods Strain and Culture Medium.--A type I strain (T1, SF130 Griflith (1)) rendered highly virulent by mouse passage was used. An 18 hour blood broth culture of this organism kills mice regularly in doses of 10- ~ to 10-8 co. For mass cultures Todd-Hewitt broth (2) prepared with neopeptone and beef heart and sterilized by filtration through porcelain filters was used without enrichment. Other media were enriched with rabbit blood. EnzymeX.--A crystalline preparation of ribonuclease, free of proteolytic activity, was used in the purification procedures. Elearophoretie Technique.--The electrophoretic studies were carried out at 0.5°C. in the apparatus described by Longsworth (3). Prior to electrophoresis a 1 per cent solution of the protein was dialyzed at 5°C. for 2 to 3 days against large volumes of the same buffe