Effect of boronic acids on antifreeze proteins.

The activity of antifreeze glycoprotein from the blood serum of Boreagadus saida was strongly inhibited by ions of organic boronic acids as well as by borate. The activity of nonglycoprotein from the blood serum of Pseudopleuronectus americanus, however, was not similarly inhibited. The inhibition by borate is thus specific for molecules with the carbohydrate moiety.

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins.

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (alpha-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was greater than 70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)2, 88; (Gly)3, 82; (Gly)4, 70; Gly-Gly-NH2, 44; Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.

Egg-white and blood-serum proteins functioning by noncovalent interactions: studies by chemical modification and comparative biochemistry.

Some of the more interesting and important proteins are those that function by forming associations or complexes with other substances. The structure-function relationships of three of these with very different substances are transferrins, which chelate metal ions; avian ovomucoids, which form complexes with proteolytic enzymes; and antifreeze glycoproteins, which interact at the ice-solution interface. Interrelating studies on the comparative biochemistry with studies using chemical modification have helped identify the side-chain groups of the proteins involved in function as well as to be useful for studies on general protein chemistry. The most strongly associated interaction is the chelation of iron by transferrin, with an association constant of approximately 10(21); tyrosines, histidines, and sometimes aspartate are involved. For ovomucoids, individual substratelike residues such as lysine are involved in a Michaelis-like complex, and association constants are as high as 10(10). By contrast, the antifreeze glycoproteins appear to function by a polymeric interaction at the surface of ice, with a much weaker association.

The interaction of anions with native and phenylglyoxal-modified human serum transferrin.

The interaction of various anions with human serum transferrin was investigated due to the concomitant binding of iron and a synergistic anion to form the transferrin-anion-iron complex. Two tetrahedral oxyanion oxidizing agents, periodate and permanganate, were found to partially inactivate transferrin when used at equimolar ratios of oxidizing agent to protein active sites. Hypochlorite, a strong oxidizing agent with little structural similarity to periodate and permanganate, had little effect on iron-binding activity when used at similar low molar ratios of reagent to transferrin active sites. Transferrin treated with a 3:1 molar ratio of periodate or permanganate to active sites lost 74 or 67% of its iron-binding capacity, respectively. The composition of the buffer affected the extent of transferrin inactivation by periodate and permanganate; for example, the extent of inactivation by periodate was threefold greater in a borate buffer than in a phosphate buffer. Comparative oxidations in buffer systems suggest the following order of affinity of three buffer anions for the apotransferrin metal-binding center: phosphate greater than bicarbonate greater than borate. The interaction of phosphate ions with the iron-transferrin complex was also examined due to the increased susceptibility to periodate inactivation of iron-saturated transferrin in phosphate buffer (M. H. Penner, R. B. Yamasaki, D. T. Osuga, D. R. Babin, C. F. Meares, and R. E. Feeney (1983) Arch. Biochem. Biophys. 225, 740-747). The apparent destabilization of the iron-transferrin complex in phosphate buffer was found to be due to the competitive removal of iron by phosphate from the iron-protein complex. We found that phenylglyoxal-modified Fe-transferrin, with no loss of bound iron, was much more resistant to iron removal by phosphate and other competitive chelators.

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis.

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).

A kinetic description of antifreeze glycoprotein activity.

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.

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.

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.

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.

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.

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.

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.

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.

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 glycoproteins from Polar fish. Effects of freezing conditions on cooperative function.

Antifreeze glycoproteins and glycopeptides that function noncolligatively contribute one-third of the freezing temperature depression in the blood serum of some polar fishes and enable them to survive at low temperature (-1.9 degree C). There are at least eight closely related glycoproteins and glycopeptides ranging in molecular weight from 32,000 to 2,600 and numbered 1 to 8 in order of decreasing size. Under conditions of negligible supercooling, the glycopeptides have weaker antifreeze activity than the glycoproteins (20% on a weight basis, or 5% on a molar basis); in mixtures of both, their activities are additive. When nucleation is initiated in supercooled solutions (-4 to -5 degrees C), the glycopeptides are inactive, while the glycoproteins still show activity; when mixtures of both are nucleated in supercooled solutions, cooperative potentiation occurs, and the full activities of the glycopeptides are found. On nucleation of supercooled solutions of the glycoprotein alone or of the mixtures, the temperature rises above the freezing temperature ("overshoots") to an extent dependent upon the extent of supercooling; the temperature of the sample then decreases to form a plateau at the true freezing temperature.

Cooperative functioning between antifreeze glycoproteins.

Antifreeze glycoproteins from polar fish bloods are a mixture of closely related components which differ structurally by size and by the presence of proline in the smaller components. Although the smaller components containing proline exist in higher amounts than do the larger ones, their presence puzzled investigators because they had very weak antifreeze activity. A very important function for these smaller components has now been found. These smaller antifreeze glycoproteins (10 to 25 mg/ml) have now been tested as mixtures with the larger active antifreeze glycoproteins (2 to 4 mg/ml) and a very large (2- to 8-fold) potentiation of antifreeze activity has been observed. There appears to be a cooperative functioning between the larger and smaller components.