David T. Osuga

The sulfhydryls of avian ovalbumins, bovine β-loctoglobulin, and bovine serum albumin☆

Abstract The sulfhydryl groups in chicken ovalbumin, bovine β-lactoglobulin, and bovine serum albumin were studied with four different types of chemical reagents: p-chloromercuribenzoate, iodine, N-ethylmaleimide and 5,5′-dithiobis (2-nitrobenzoic acid). Native chicken ovalbumin had three weakly reactive (“masked”) SH groups, and after denaturation it had four reactive groups. Native β-lactoglobulin had no, or only very weakly, reactive groups; after denaturation it had two reactive groups. Serum albumin gave similar values before and after denaturation, but the values of different preparations varied between 0.3 and 0.75 SH groups. In comparative analyses of the SH contents of the egg whites of 22 different avian species, only one other bird besides the domestic chicken showed additional SH groups after denaturation. This bird was the red jungle fowl, which is very closely related to the domestic chicken. The 5,5′-dithiobis (2-nitrobenzoic acid) was considered the most useful reagent for routine determinations of the approximate total content of SH groups.

Evolution of flightless land birds on southern continents: Transferrin comparison shows monophyletic origin of ratites

SummaryA biochemical approach was used to study the evolution of ratite birds, i.e., the ostriches, rheas, cassowaries, emus, and kiwis. Quantitative immunological comparison of transferrin from ratites, tinamous, and other flying birds indicates that all the ratites and tinamous are allied phylogenetically and that they are of monophyletic origin relative to other birds. To explain the current geographic distribution of ratites and the magnitude of the transferrin distances, it is supposed that the ancestors of these flightless birds walked across land bridges between the southern continents during Cretaceous times.

Penguin evolution: protein comparisons demonstrate phylogenetic relationship to flying aquatic birds.

SummaryQuantitative immunological comparisons of three avian proteins, transferrin, ovalbumin, and penalbumin, indicate that penguins are phylogenetically most closely related to loons, albatrosses, herons, and grebes. These data support the theory that the ancestors of penguins were flying oceanic birds and that flightlessness in penguins has evolved independently from flightlessness in ratites.

Determination of hydrogen sulfide with 5,5′-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, and parachloromercuribenzoate

Hydrogen sulfide was determined with N-ethylmaleimide (NEM), p-chloromercuribenzoate (PCMB), and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). One mole of hydrogen sulfide reacted with two moles of NEM or PCMB. It reacted with only one mole of DTNB, but produced two moles of the thiol anion, 5-thio-2-nitrobenzoate, and one mole of free sulfur. Concentrations of hydrogen sulfide down to 3.7 × 10−6m (0.13 μg/ml) could be easily determined with DTNB. In the presence of proteins containing different amounts of sulfhydryls reacting with DTNB, 78 to 94% of the added hydrogen sulfide could be recovered.

Biochemistry of the egg-white proteins of the ratite group.

Abstract The properties of the avian egg-white proteins from the five living members of the ratite group and one related species were studied. The ratite egg whites examined were cassowary ( Casuarius aruensis ), emu ( Dromiceius n. hollandiae ), kiwi ( Apteryx mantelli ), ostrich ( Struthio camelus ), and rhea ( Rhea americana ). The egg white of the related species was that of the tinamou ( Eudromia elegans ). Examinations included starch and acrylamide gel electrophoreses of whole egg whites; direct determinations for the amounts of biochemically detectable constituents in whole egg whites; physical and chemical separations of individual constituents; and the examinations of properties of purified fractions. The lysozymes, ovomucoids, ovotransferrins, ovoflavoproteins, ovomucins, ovalbumins, and components containing sialic acid and amlyase activity were examined. Comparisons were also made with the egg whites and egg-white constituents of the chicken ( Gallus gallus ) and the turkey ( Maleagris gallopavo ). Many of the individual constituents had similar physical and chemical properties, but definite differences existed in most cases. The most obvious differences were in the specificities of the ovomucoids against trypsin and chymotrypsin, a different activity of kiwi lysozyme, the presence of varying amounts of a constituent with amylase activity, and the presence of either comparatively low or high amounts of sialic acid. Close biochemical and immunochemical relationships were found among the ratites, and they appeared remotely related to the tinamou.

Heterogeneity of avian ovomucoids

Abstract Studies were made on the heterogeneity of avian ovomucoids in preparations from eggs of different breeds and genetic lines of chickens and from eggs of different avian species. The ovomucoids from eggs of only two of a total of eleven different strains or breeds of chickens showed definite differences in electrophoretic patterns. The other nine were indistinguishable and all eleven were indistinguishable based on other physical and chemical properties examined. Preparations of ovomucoids of ten other avian species were also found to be heterogeneous electrophoretically as examined in starch and polyacrylamide gels. The ovomucoids of most of the species gave electrophoretic patterns different from one another. Ovomucoids from single eggs of the two distantly related species, the chicken and the cassowary, were heterogeneous. Different fractions of cassowary ovomucoid, which were separated by ion-exchange chromatography, gave different electrophoretic patterns and showed differences in sialic acid ranging from approximately 2 to 5%. Heterogeneity of avian ovomucoids thus appears to be a general phenomenon in the avian egg whites of all species.

Periodate oxidation of methionine in proteins.

Abstract Treatment of free methionine with an equimolar amount of periodate gave nearly quantitative formation of the sulfoxide; treatment of free methionine sulfoxide with equimolar periodate gave nearly equal amounts of the original sulfoxide and the sulfone. Treatments of 0.5-1.0% solutions of the following proteins with relatively low concentration of periodate (5 m m ) gave the following approximate values for conversion of methionine sulfoxide from total methionine: bovine pancreatic ribonuclease A (2 of 4), chicken ovalbumin (14 or 17), chicken ovotransferrin (5 of 11), human serum transferrin (2 of 8), bovine α-chymotrypsin (1 of 2). It is recommended that when proteins are treated with sodium periodate (and probably with oxidizing agents in general), especially when changes in properties are observed, determinations of methionine sulfoxide should be done.

Pyridine borane as a reducing agent for proteins

Pyridine borane has been reported as a superior reagent over a wide pH range, 5-9, for the reductive methylation of amino groups of proteins with formaldehyde [J. C. Cabacungan , A. I. Ahmed , and R. E. Feeney (1982) Anal. Biochem. 124, 272-278]. It has also been reported to reduce tryptophan to dihydrotryptophan and to inactivate lysozyme in trifluoroacetic acid [M. Kurata , Y. Kikugawa , T. Kuwae , I. Koyama , and T. Takagi (1980) Chem. Pharm . Bull 28, 2274-2275]. In the present study the specificity of pyridine borane for the two different modifications under different reaction conditions has been demonstrated, and extended to the application to the synthesis of protein containing reductively attached carbohydrates. In the acid reduction, pyridine borane selectively reduced all six tryptophans in lysozyme to dihydrotryptophan while all other amino acids remained intact. On similar treatment no cleavage of the carbohydrate moiety from chicken ovomucoid, and no losses of activity of ovomucoid or ribonuclease, two proteins devoid of tryptophan, were observed. Nearly complete methylation of the lysines of lysozyme, chicken ovomucoid, and ribonuclease was achieved with formaldehyde at pH 7.0 after 2 h at room temperature, with the retention of full activity of the protein without any destruction of tryptophan. The same chemistry was applied to covalently attach glucose and lactose to bovine serum albumin. Parameters, including pH, temperature, and methanol, that affect the reactions were investigated. Incremental additions of pyridine borane during the course of the reactions increased the rate of modification. The covalent attachment of sugar to the epsilon-amino group of lysine was demonstrated by the synthesis of N-alpha- acetylglucitollysine and comparison with acid hydrolysates of the bovine serum albumin-sugar derivatives.

Blood plasma proteins of cold-adapted antarctic fishes☆

Abstract 1. 1. All fish serum transferrins studied were more acidic than human serum transferrin. 2. 2. Trematomus borchgrevinki serum transferrin was more heat labile than human serum transferrin. 3. 3. Antarctic fish serum had less albumin and more lipoprotein than sera of non-Antarctic fishes and of man, and contained freezing-point-depressing glycoproteins which were absent in all other sera studied. 4. 4. The bloods of two cold-adapted fishes, T. borchgrevinki and trout, clotted faster at 0°C than did the blood of two other non-cold-adapted fish species studied. Bloods of the two mammalian species studied, rabbit and man, had very prologned clotting times at 0°C. 5. 5. The blood of T. borchgrevinki had an apparent minimum clotting time between 25 and 30°C and its capacity to clot was destroyed by incubation at 38–40°C.

Synthesis of immobilized flavin derivatives and their use in purification of chicken egg-white ovoflavoprotein

Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the flavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.