Abstract
We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aisen P., Leibman A., Zweier J. Stoichiometric and site characteristics of the binding of iron to human transferrin. J Biol Chem. 1978 Mar 25;253(6):1930–1937. [PubMed] [Google Scholar]
- Aisen P., Listowsky I. Iron transport and storage proteins. Annu Rev Biochem. 1980;49:357–393. doi: 10.1146/annurev.bi.49.070180.002041. [DOI] [PubMed] [Google Scholar]
- Bates G. W., Schlabach M. R. The reaction of ferric salts with transferrin. J Biol Chem. 1973 May 10;248(9):3228–3232. [PubMed] [Google Scholar]
- Chasteen N. D. Transferrin: a perspective. Adv Inorg Biochem. 1983;5:201–233. [PubMed] [Google Scholar]
- Chasteen N. D., Williams J. The influence of pH on the equilibrium distribution of iron between the metal-binding sites of human transferrin. Biochem J. 1981 Mar 1;193(3):717–727. doi: 10.1042/bj1930717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Constans J., Kühnl P., Viau M., Spielmann W. A new procedure for the determination of transferrin C (Tf C) subtypes by isoelectric focusing. Existence of two additional alleles, Tf C4 and Tf C5. Hum Genet. 1980;55(1):111–114. doi: 10.1007/BF00329135. [DOI] [PubMed] [Google Scholar]
- D'Alessandro A. M., D'Andrea G., Oratore A. Different patterns of human serum transferrin on isoelectric focusing using synthetic carrier ampholytes or immobilized pH gradients. Electrophoresis. 1988 Feb;9(2):80–83. doi: 10.1002/elps.1150090204. [DOI] [PubMed] [Google Scholar]
- Hovanessian A. G., Awdeh Z. L. Gel isoelectric focusing of human-serum transferrin. Eur J Biochem. 1976 Sep 15;68(2):333–338. doi: 10.1111/j.1432-1033.1976.tb10819.x. [DOI] [PubMed] [Google Scholar]
- Lineback-Zins J., Brew K. Preparation and characterization of an NH2-terminal fragment of human serum transferrin containing a single iron-binding site. J Biol Chem. 1980 Jan 25;255(2):708–713. [PubMed] [Google Scholar]
- Makey D. G., Seal U. S. The detection of four molecular forms of human transferrin during the iron binding process. Biochim Biophys Acta. 1976 Nov 26;453(1):250–256. doi: 10.1016/0005-2795(76)90270-1. [DOI] [PubMed] [Google Scholar]
- Zak O., Aisen P. Preparation and properties of a single-sited fragment from the C-terminal domain of human transferrin. Biochim Biophys Acta. 1985 Jul 1;829(3):348–353. doi: 10.1016/0167-4838(85)90243-2. [DOI] [PubMed] [Google Scholar]