Search PubMed⌕ Search

Biomedical subjects

R E Feeney

Publications and source records attributed to R E Feeney.

At least 37 records · Page 2Linked to original sources
PubMed1986

Antifreeze glycoproteins from polar fish blood.

Existing experimental evidence strongly suggests that the mechanism of activity of the Antarctic AFGP molecules is the inhibition of ice growth by competitive adsorption onto the growth sites of ice. The data further suggest the blocking of the formation of large critical nuclei for ice growth. Experiments showing that the longer polymers (AFGP 1-5) have different growth-prevention properties with different types of ice than the shorter polymers (AFGP 6-8) provide additional evidence that crystal size and habits are linked to function. Four main observations have been used in AFGP studies: (a) The ice crystal habit (size) affects the activity, (b) AFGP is on the surface of ice crystals, as shown by surface second harmonic generation (SSHG), (c) the presence of AFGP lowers the surface energy at the ice-solution interface, and (d) kinetic calculations of the inhibition of ice-crystal growth fit adsorption isotherms. In particular, AFGP 4 fits a Langmuirian adsorption curve. On the detailed mechanistic side, there is a need to quantify the competitive rates of water-molecule attachment to ice as well as the rates of adsorption-desorption of AFGP on the ice surface. Toward this end, experiments are currently being conducted to examine the differential growth rates of ice crystals freely growing into a solution of AFGP. Direct observation of the adsorption and desorption of AFGP on the ice surface is possible in principle using the SSHG technique. A nonperturbative probe method that can differentiate adsorbed AFGP from the solution phase molecules is needed. Finally, a mechanistic description of molecular function is never complete unless the detailed molecular binding to the surface is elucidated. This task has not been accomplished yet, and thus there is still a question as to which molecular group is actually adsorbed onto the surface and for how long. Theoretically, as we have seen, the departure from Langmuirian adsorption is pronounced for small AFGP molecules under certain conditions. More refinements of the ideas of intermolecular or intramolecular cooperative interaction of AFGP on the ice surface are needed.

Adsorption↗
PubMed1985

Interaction of oxidized chicken ovotransferrin with chicken embryo red blood cells.

Iron-saturated chicken ovotransferrin was chemically oxidized with NaIO4, converting 50% of its methionine residues to their sulfoxide derivatives while maintaining 95% of its iron-binding activity. The oxidized chicken ovotransferrin was able to deliver iron to the chicken embryo red blood cell for heme synthesis. From competition experiments, oxidized diferric chicken ovotransferrin was estimated to be approx. 65% as efficient as unmodified diferric chicken ovotransferrin at competing with diferric (55Fe2) chicken ovotransferrin for the iron-donating sites of the chicken embryo red blood cells. The presence of apo chicken ovotransferrin preparations (native or oxidized) in the incubation medium had little effect on the rate of iron incorporation into heme from diferric chicken ovotransferrin. The effect of modifying the periodate-susceptible methionine residues in chicken ovotransferrin was small but significant. These methionine residues do not appear critical for the interaction of chicken ovotransferrin with the chicken embryo red blood cell receptors, the incorporation of chicken ovotransferrin into the cell, or the release of iron from chicken ovotransferrin for heme synthesis.

Animals↗
PubMed1985

1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation.

Reductive alkylation of lysyl epsilon-amino groups with sugars (1-deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58-67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1-deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity.

Animals↗
PubMed1984

Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography.

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.

Amino Acids↗
PubMed1984

Conformation of the antifreeze glycoprotein of polar fish.

High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order.

Animals↗
PubMed1984

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.

Alkylation↗
PubMed1984

Analysis of antifreeze glycoproteins in fish serum.

A procedure utilizing high-pressure size-exclusion chromatography that permits rapid screening for both the types of components present in and the quantity of antifreeze glycoprotein in fish serum or solution is described. The applicability of the method is demonstrated by a comparative study of five different fish species, four of which contain the antifreeze glycoprotein and one which does not contain this protein. The antifreeze glycoprotein compositions of two fish of the same species, collected at different locations or under different environmental conditions, are also compared. A linear molecular-weight versus elution-volume function is established for both standard native proteins and the antifreeze glycoproteins, but these two lines do not coincide. The differences in tertiary structure between the antifreeze glycoproteins and normal proteins are presented as an explanation for the nonequivalence of calibration lines.

Animals↗
PubMed1983

Comparative oxidations of tyrosines and methionines in transferrins: human serum transferrin, human lactotransferrin, and chicken ovotransferrin.

Periodate treatments of apo human serum transferrin (HST), and apo chicken ovotransferrin (COT) were previously reported to cause a rapid loss of Fe+3 binding capacity, with a loss of 3 to 5 tyrosine residues [P. AZARI AND J. L. PHILLIPS (1970) Arch. Biochem. Biophys. 138, 32-38; K. F. GEOGHEGAN, J. L. DALLAS, AND R. E. FEENEY (1980) J. Biol. Chem. 255, 11429-11434]. The effects of periodate and hydrogen peroxide on human lactotransferrin (HLT), HST, and COT have been compared. All three apotransferrins were rapidly inactivated and lost approximately 4 to 5 tyrosine residues by 5 mM periodate treatment; their iron complexes had little or no inactivation and losses of approximately 1 to 2 tyrosine residues. All three iron transferrins were highly resistant to inactivation by 5 mM periodate in bicarbonate, with or without the addition of phosphate, while in phosphate (with ambient carbonate) Fe2HLT was highly resistant, Fe2COT slightly less resistant, and Fe2HST much less resistant. Similar oxidations of methionines to the sulfoxides were found in both the apo and iron forms. After 150 min of 5 mM periodate treatment HST lost approximately 3 (apo 3.1, iron 2.8) of 9, HLT approximately 3 (apo 2.6, iron 2.9) of 6, and COT approximately 7 (apo 7.2, iron 7.2) of 11 methionines per mole of protein. In the presence of 8 M urea HST had essentially all of its methionine residues oxidized by periodate, but only lost part of its activity on renaturation. Treatment of all apo transferrins with 300 mM hydrogen peroxide resulted in little or no losses (less than 10%) in activity. HST lost approximately one-third of its methionines and no tyrosines during the 300 mM hydrogen peroxide treatment. Therefore the essentiality of tyrosines for all three transferrins was confirmed and the nonessentiality of methionines was demonstrated.

Amino Acids↗
PubMed1983

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.

Amides↗
PubMed1982

Penguin egg-white and polar fish blood-serum proteins.

The development of, and findings in, a long-term research program on penguin proteins and polar fish blood proteins are described. Two of the egg-white proteins from the Adelie penguin (Pygoscelis adeliae) have unique properties: a glycoprotein named penalbumin that is a major constituent with some characteristics similar to ovalbumin, and an ovomucoid with strong inhibitory capacity for subtilisin as well as for bovine trypsin and alpha-chymotrypsin. The antifreeze glycoproteins from Antarctic fish (Trematomus borchgrevinki and Dissostichus mawsoni) and an Arctic fish (Boreogadus saida) appear to function noncolligatively by lowering the freezing temperature without affecting the melting point. Current evidence indicates that the antifreeze glycoprotein functions at the ice-solution interface, either on the ice surface or in a transition layer between the solution and the ice.

Animals↗
PubMed1981

Antifreeze glycoprotein. Conformational model based on vacuum ultraviolet circular dichroism data.

Circular dichroism spectra (CD) of an active fraction of antifreeze glycoprotein (AFGP) and of a lower molecular weight fraction which is less active are found to be similar in the wavelength range 170-230 nm. The contribution of the disaccharide side chain chromophores to the CD spectra of the glycoprotein is estimated from spectra of model oligosaccharides having similar structures. Comparison of the CD spectra of the peptide portion of AFGP to those of established model polypeptides suggests a 3-fold left-handed helix of the collagen type as the most likely conformation of the peptide. In the proposed model, the disaccharide moieties attached to every third residue are in identical environments. Furthermore, proline residues which are found in the less active fraction can be accommodated in this helical structure, consistent with the observed similarity of the spectra of the two fractions.

Antifreeze Proteins↗
PubMed1981

Alternative reducing agents for reductive methylation of amino groups in proteins.

Dimethylamine borane and trimethylamine borane were compared with sodium cyanoborohydride as reducing agents for the reductive methylation of amino groups in proteins. Dimethylamine borane performed nearly as well as cyanoborohydride; either reducing agent (15 mM) with 20 mM formaldehyde gave extensive methylation of turkey ovomucoid (5 mg/ml) in 2 h at 22 degrees. Trimethylamine borane gave equivalent levels of methylation only with a higher concentration of formaldehyde, suggesting that it is an even milder reducing agent than the other two under these conditions. With all three reducing agents, the pH optimum for methylation covered a range of pH 6-9. Methylations should be performed at the lowest possible pH to avoid side reactions of formaldehyde with the protein. Possible effects of these reducing agents on the disulfide bonds of proteins were studied. No reduction of disulfides of turkey ovomucoid was observed using each of the three reducing agents under conditions for methylation.

Amino Acids↗
PubMed1980

Periodate inactivation of ovotransferrin and human serum transferrin.

Azari and Phillips (Azari, P., and Phillips, J. L. 1970 Arch. Biochem. Biophys. 138, 32-38) reported that periodate treatment of iron-free ovotransferrin causes a rapid loss of iron-binding activity and an oxidation of 3 to 5 tyrosines and 1 tryptophan. Rapid inactivation and loss of tyrosine in ovotransferrin has been confirmed, and the work extended to human serum transferrin and effects of denaturing concentrations of urea. Extensive (> 80%) inactivations of both ovotransferrin and human serum transferrin were observed when approximately 4 tyrosines were destroyed. Amino acid analysis and 360-MHz 1H NMR spectra confirmed that tyrosines are the only residues rapidly oxidized; the correlation of tyrosine loss with the loss of iron-binding activity suggests strongly that the tyrosines involved are those that function as ligands to metal ions bound to the protein. NMR spectra also showed that periodate oxidation causes local changes of structure in ovotransferrin (presumably at the metal-binding sites) but does not grossly alter the conformation. The addition of 5 to 8 M urea greatly retarded the inactivation and losses of tyrosine.

Amino Acids↗