Preparation of Amino-Terminal Half-Molecule of Ovotransferrin by Tryptic Digestion of Intact Molecule Selectively Saturated with Al(III) at the<i>N</i>-Lobe

Nakazato, Ken’ichiro; Enami, Isao; Tanaka, Yuichi; Uchiyama, Yotaro; Tsugita, Akira; Satake, Kazuo

doi:10.1271/bbb.56.687

Cited by 5 publications

(3 citation statements)

References 2 publications

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“…Human apo-sTf and chicken apo-OTf were purchased from Sigma Chemical Co. and used without further purification. The Nand C-terminal half-molecules of OTf (OTf/2N and OTf/2C) were isolated from the mild digestion of diferric OTf (Sigma Chemical Co.) with TPCK-treated trypsin and characterized following published procedures (Oe et al, 1988;Nakazato et al, 1992;Thornton et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Thallium-205 and Carbon-13 NMR Studies of Human Sero- and Chicken Ovotransferrin

Aramini¹,

Krygsman²,

Vogel³

1994

Biochemistry

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We have examined the binding of Tl3+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by 205Tl and 13C NMR spectroscopy. With carbonate as the synergistic anion, one observes two 205Tl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of each protein. When the same adducts are prepared with 13C-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the 13C NMR spectrum of serotransferrin; these correspond to the labeled anion directly bound to the metal ion in both sites of the protein. The analogous resonances in ovotransferrin are completely degenerate, and only one doublet can be detected. The magnitudes of the spin-spin coupling between the bound metal ion and carbonate range from 2J(205Tl-13C) approximately 270 to 290 Hz. We have used the proteolytic half-molecules of ovotransferrin and the recombinant N-terminal half-molecule of serotransferrin to assign the 205Tl and 13C NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that Tl3+ is bound with a greater affinity at the C-terminal site of serotransferrin, whereas no site preference can be noted for ovotransferrin. When oxalate is used as the anion instead of carbonate, the 205Tl NMR signals arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex pattern of resonances is observed for bound 13C2O4(2-) in the 13C NMR spectra of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Thallium-205 and Carbon-13 NMR Studies of Human Sero- and Chicken Ovotransferrin

Aramini¹,

Krygsman²,

Vogel³

1994

Biochemistry

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“…Briefly, iron-saturated OTf was partially digested with trypsin and the nicked-OTf was purified by Sephadex G-100 and DEAE-Sephadex A-50 columns followed by CM-Sephadex C-50 column to resolve the N-and C-lobes [27]. To remove iron, the individual lobes were dialyzed first against 0.1 M citrate (pH 4.5) containing 0.1% EDTA, then against an excess of Milli Q water for 2 days and freeze dried.…”

Section: Preparation Of the N-and C-lobes Of Otfmentioning

confidence: 99%

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

Ibrahim

Hoq

Aoki

2007

International Journal of Biological Macromolecules

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“…Moreover, to investigate the enzymatic activity of lysozyme within the complex and after release from the complex, it was necessary to selectively digest and remove the constituent proteins of the complex. Egg white contains proteins like ovalbumin and ovotransferrin that are digested by trypsin, and other proteins that are not digested by trypsin [17–19]. It has been reported that the interaction between lysozyme and other proteins like ovotransferrin reduces enzymatic activity of lysozyme [20], and enzymatic activity of lysozyme within the lysozyme–ovotransferrin complex is believed to change after digestion and removal of ovotransferrin using trypsin.…”

Section: Introductionmentioning

confidence: 99%

Characterization of enzymatic activity of lysozyme in lysozyme–ovotransferrin complex before and after treatment with trypsin

Shimazaki

Yabu

2021

Separation Science Plus

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The native complex of lysozyme and ovotransferrin was isolated after the separation of egg white proteins using non‐denaturing isoelectric focusing, and mobilization toward the cathode by replacement of the cathodic sodium hydroxide solution with a phosphoric acid solution. The treatment of the lysozyme–ovotransferrin complex with trypsin significantly increased the enzymatic activity of lysozyme. Likewise, an increase in the enzymatic activity of lysozyme was also obtained when a mixture of the purified lysozyme and ovotransferrin was treated with trypsin. The increase in lysozyme enzymatic activity after tryptic treatment of the lysozyme–ovotransferrin complex resulted from the liberation of lysozyme from this complex. This was a consequence of the resistance of lysozyme to tryptic digestion and the digestion of ovotransferrin by trypsin into peptide fragments, which do not bind the lysozyme. The developed methodology could be used for the separation and isolation of other protein complexes and for testing their enzymatic and other activities.

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Preparation of Amino-Terminal Half-Molecule of Ovotransferrin by Tryptic Digestion of Intact Molecule Selectively Saturated with Al(III) at theN-Lobe

Cited by 5 publications

References 2 publications

Thallium-205 and Carbon-13 NMR Studies of Human Sero- and Chicken Ovotransferrin

Thallium-205 and Carbon-13 NMR Studies of Human Sero- and Chicken Ovotransferrin

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

Characterization of enzymatic activity of lysozyme in lysozyme–ovotransferrin complex before and after treatment with trypsin

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