Preparation and Characterization of an NHz-terminal Fragment of Human Serum Transferrin Containing a Single Iron-binding Site*

نویسنده

  • Keith Brew
چکیده

A fragment of M, = 35,600 containing a single ironbinding site has been prepared from diferric human serum transferrin by digestion with thermolysin at 37°C and purified by gel filtration. Prolonged digestion led to greater hydrolysis of the diferric protein and lower yields of the fragment. However, a similar ratio of fragment to uncleaved protein was obtained at all incubation times suggesting that the fragment represents a stable intermediate in the digestion of the protein. Similar yields of fragment were obtained when digestion was carried out at pH values between 6.1 and 7.5. The fragment is devoid of carbohydrate. Following CNBr (cyanogen bromide) cleavage, fragments corresponding to those present in the NH2-terminal region of transferrin (CN-6, CN-5, CN-2, and CN-7) were isolated and identified. An additional fragment, characterized by amino acid composition and sequence analysis, was obtained which consists of part of a CNBr fragment (CN-4) of transferrin that spans the region of the molecule between the two homologous domains. From this, the site of cleavage between the NH2and COOH-terminal domains was identified as a Pro-Val bond within CN-4. Internal cleavages within the fragment were identified, one resulting in the loss of a tyrosine-rich region near the COOH terminus of the domain, and another being the cleavage of a Tyr-Leu bond between residues 7 1 and 72. Studies of the pH stability of the metal-binding site of the fragment suggest that it may correspond to a site which is stable at pH 6.0.

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Preparation and characterization of an NH2-terminal fragment of human serum transferrin containing a single iron-binding site.

A fragment of M, = 35,600 containing a single ironbinding site has been prepared from diferric human serum transferrin by digestion with thermolysin at 37°C and purified by gel filtration. Prolonged digestion led to greater hydrolysis of the diferric protein and lower yields of the fragment. However, a similar ratio of fragment to uncleaved protein was obtained at all incubation times suggestin...

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تاریخ انتشار 2001