Robert E. Feeney

Carbonyl-Amine Reactions in Protein Chemistry

Publisher Summary Reaction of a carbonyl group with an amino group is the key reaction in many enzymic and other biological processes, such as vision. This chapter discusses and attempts to organize and correlate the different types of carbonyl–amine reactions found in proteins with their organic chemistry. The chapter focuses on the carbonyl–-amine reaction in biological processes, naturally occurring deteriorative reactions, and their commercial applications. The most characteristic chemical property of amines is their ability to act as nucleophiles because they possess a lone pair of electrons on the nitrogen atom, while, the reactivity of the carbonyl bond is primarily because of the difference in electronegativity between carbon and oxygen, which leads to a significant contribution of the dipolar resonance form, oxygen being negative and carbon being positive. The rate of the carbonyl–amine reaction usually shows acharacteristic pH dependence that results in a bell-shaped curve. Formaldehyde reacts not only with primary amino groups in proteins, but also with sulfhydryl groups. Carbonyl–amine reactions play important roles in catalytic reactions in enzymes, cross-linking in structural proteins like collagen and elastin and the visual process.

Antifreeze proteins from fish bloods.

Publisher Summary Glycoproteins of molecular weights isolated from the blood sera of the two Antarctic fishes, Trematomus borchgrevinki and Dissostichus mawsoni, exhibit antifreezing properties in pure H2O or salt solutions. As the melting point is not affected, a hysteresis exists between the freezing and melting temperatures, and the glycoproteins are considered to lower the freezing temperature rather than the freezing point. The information obtained on the basic structure of these glycoproteins is shared, and models of antifreeze mechanism are discussed. In these models, the polypeptide is assumed to form a nonideal, nonregular solution in H20. In one model, the ability of the hydrophilic sites of these molecules to enter into favorable competition for hydrogen bonds among the H20 molecules themselves provides for a mechanism of lowering the freezing point. The only over-riding similarity amongthese antifreezing molecules is the predominance of alanine. It is this feature that has primary and critical importance in antifreeze function observed in these fishes. Antifreeze glycoprotein (AFGP) discussed is a collective name for a family of at least eight closely related glycoproteins that account for a major fraction of the protein in the blood serum of certain Antarctic fishes the eight originally observed glycoproteins during electrophoresis in acrylamide gel with borate buffer. Along with lowering the freezing temperature of water to an abnormal extent as based on their particle sizes, active AFGP components also are abnormal in several other properties related to lowering the freezing temperature and the freezing process itself.

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.

Antifreeze proteins: Current status and possible food uses

Antifreeze proteins from the blood serum of cold-water ocean fish inhibit ice crystal growth, including recrystallization, and affect crystal morphology. They exist in relatively high concentrations, ∼30 mg/ml, and are peptides or small proteins, mainly with less than 40 amino acids. Structures include both glycoprotein and non-glycoproteins, some of which have been chemically synthesized. They have also been found in plants and insects. The most successful experiments on foods have been the reduction of recrystallization in frozen dairy products. Cost, however, is apparently presently blocking usage.

Chemical and physical modification of proteins by the hydroxide ion

Proteins are exposed to alkaline conditions during solubilization and/or purification, during food storage and processing, in removal of toxic constituents, and for characterization. During alkali treatment, there are changes in solubility and aggregation, hydrolysis, elimination reactions involving the side chains of certain amino acids, racemization of amino acid residues, addition of compounds to proteins, fragmentation of the peptide chain, as well as modification or elimination of nonprotein constituents. The rates of these reactions are affected by pH, temperature, cations (in some cases), ionic strength (in some cases), protein concentration, and to some extent by the specific nature of the protein. The general mechanisms and stoichiometry of these reactions are described. Other constituents of high protein foods also undergo reactions in alkaline solutions and the products of these reactions may in turn react with proteins. We have described the effect of alkali on enediol formation and fragmentation of carbohydrates, the hydrolysis of lipids in alkaline solution and effect on rate of peroxidation of the polyunsaturated fatty acids, the oxidation of amino acid residues, especially methionine, the oxidation of phenols to benzoquinones, and the catalytic effect of metal ions in alkaline solutions. Alkali treatment is also used in the specific modification of proteins to distinguish between O-glycosyl and amide-linked glycosyl groups, to effect specific cleavage of peptide bonds via beta elimination, in the formation of anhydrotrypsin, anhydrochymotrypsin, anhydrosubtilisin and thiol-subtilisin, and in formation of intrachain crosslinking in proteins.

Modification of available arginine residues in proteins by p-hydroxyphenylglyoxal.

Abstract p -Hydroxyphenylglyoxal reacts with arginine residues in proteins to give a single product which can be quantitated spectrophotometrically. The reaction takes place under mild conditions, pH 7–9 and 25°C. Under these conditions up to complete modification of N α -citraconyl- l -arginine was obtained within 60 min with less than 5% modification of other common amino acid side chains. The extent of modification in a protein can be determined at 340 nm using the molar absorption coefficient of 1.83 × 10 4 m −1 cm −1 for the product at pH 9.0 and 25°C following removal of excess reagent by gel filtration. Several proteins, previously shown to have essential arginines, were modified by p -hydroxyphenylglyoxal and the losses in arginines were determined spectrophotometrically. These results were in close agreement with those of previous investigators. Rhea ovomucoid, a glycoprotein without arginines but containing an essential lysine, was relatively unaffected.

A mechanism for stabilization of membranes at low temperatures by an antifreeze protein

Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.

Amine boranes as alternative reducing agents for reductive alkylation of proteins

Abstract The use of several commercially available amine-borane complexes was investigated for the reductive methylation of amino groups of several proteins. An earlier study in our laboratory, using turkey ovomucoid as the model protein, showed that dimethylamine borane is a slightly weaker reducing agent, but a good, less toxic substitute for sodium cyanoborohydride ( K. F. Geoghegan, J. C. Cabacungan, H. B. F. Dixon, and R. E. Feeney, 1981 , Int. J. Peptide Protein Res. 17 , 345–352). N -α-Acetyl- l -lysine, poly- l -lysine, turkey ovomucoid, bovine serum albumin, chicken ovalbumin, β-lactoglobulin, casein, and soybean protein were reductively methylated with dimethylamine borane and trimethylamine borane. The latter produced a consistently lower degree of modification even in the presence of sodium dodecyl sulfate. In a comparison that included the boranes triethylamine, t-butylamine, morpholine, and pyridine, pyridine borane was found to be slightly stronger than sodium cyanoborohydride. In a pH 7 solution containing 2 m m N -α-acetyl- l -lysine and 20 m m formaldehyde, complete dimethylation was achieved with about 10 m m pyridine borane after 2 h incubation at 22°C, while more than 15 m m was necessary with sodium cyanoborohydride. Like dimethylamine borane, both pyridine borane and triethylamine borane showed a reducing capacity at pH 7 which was as high as that at pH 9. Reductive alkylation under neutral to mild acid conditions allows modification of alkaline labile proteins and also limits the side reactions between proteins and formaldehyde.

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.

The sulfhydryl groups of ovalbumin

Abstract 1. 1. The reactivity of the sulfhydryl groups of ovalbumin has been investigated by iodine oxidation studies and by a titrimetric method with p-chloromercuribenzoate. The titrimetric method involved the addition of p-chloromercuribenzoate to solutions of the protein buffered at pH 5.3 and back titration of the excess or unreacted mercury reagent with cysteine, with nitroprusside as an outside indicator. 2. 2. The derivative of native ovalbumin and p-chloromercuribenzoate was recrystallized. It was dissociated by dialysis against excess cysteine at pH 7.0 and the original protein could be recovered. 3. 3. Native ovalbumin was found to contain approximately 3 equiv./ mole reactive sulfhydryl, and denatured ovalbumin was found to contain 4 equiv./mole. 4. 4. Partial oxidation of native ovalbumin with iodine followed by p-chloromercuribenzoate titrations indicated that iodine preferentially oxidizes those sulfhydryl groups that react with p-chloromercuribenzoate in the native protein. 5. 5. It was concluded from titrations of native and denatured whole egg white that the ovalbumin contains most, if not all, of the sulfhydryl groups in egg white.