scholarly journals Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins

1996 ◽  
Vol 318 (1) ◽  
pp. 145-148 ◽  
Author(s):  
Qing-Yu HE ◽  
Anne B MASON ◽  
Robert C WOODWORTH
Keyword(s):  
Metal Binding ◽  
Single Point ◽  
Spectrophotometric Titration ◽  
Serum Transferrin ◽  
Binding Properties ◽  
Break Points ◽  
Recombinant Technology ◽  
Binding Avidity ◽  
Spectral Titration

A rapid and sensitive technique, involving difference spectral titration with cobalt(III), to measure the ϵ values of chicken ovotransferrin, human serum transferrin, the N-lobe of human transferrin and several single point mutants is reported. The resulting ϵ values were compared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411–2423]. The titrations with cobalt feature sharp break-points and do not destroy the protein samples. The choice of buffer was found to be important, depending on the metal-binding avidity of the proteins. Cobalt titration should prove useful for studying the comparative metal-binding properties of transferrin and mutants of transferrin being generated by recombinant technology.

scholarly journals Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe

2001 ◽  
Vol 354 (2) ◽  
pp. 423 ◽  
Author(s):  
Qing-Yu HE ◽  
Anne B. MASON ◽  
Barbara A. LYONS ◽  
Beatrice M. TAM ◽  
Vinh NGUYEN ◽  
...  
Keyword(s):  
Metal Binding ◽  
Single Point ◽  
Binding Properties ◽  
Human Transferrin

Effects of Mutations of Aspartic Acid 63 on the Metal-Binding Properties of the Recombinant N-Lobe of Human Serum Transferrin†

Biochemistry ◽  
1997 ◽  
Vol 36 (18) ◽  
pp. 5522-5528 ◽  
Author(s):  
Qing-Yu He ◽  
Anne B. Mason ◽  
Robert C. Woodworth ◽  
Beatrice M. Tam ◽  
Toby Wadsworth ◽  
...  
Keyword(s):  
Human Serum ◽  
Aspartic Acid ◽  
Metal Binding ◽  
Serum Transferrin ◽  
Binding Properties ◽  
Human Serum Transferrin

Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe

2001 ◽  
Vol 354 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Qing-Yu HE ◽  
Anne B. MASON ◽  
Barbara A. LYONS ◽  
Beatrice M. TAM ◽  
Vinh NGUYEN ◽  
...  
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Fluorescence Spectra ◽  
Present Report ◽  
Single Point ◽  
Tryptophan Residue ◽  
Iron Binding ◽  
Iron Release ◽  
Binding Properties ◽  
Tryptophan Residues

Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp8, Trp128 and Trp264, located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV–visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp8 resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two β-sheets containing Trp8 and Lys296 respectively. The fluorescence of Trp8 may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp8→Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp128 results from the nearby His119 residue. Difference-fluorescence spectra reveal that any protein containing Trp128 shows a blue shift upon binding metal ion, and the NMR signal of Trp128 broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp128 is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp264 is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp264 is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp264 has little effect on the iron-binding and release behaviours of the protein.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

Synthesis and Metal-Binding Properties of Chelating Fluorescein Derivatives

2003 ◽  
Vol 5 (12) ◽  
pp. 2051-2054 ◽  
Author(s):  
Matthew A. Clark ◽  
Kathryn Duffy ◽  
Jyoti Tibrewala ◽  
Stephen J. Lippard
Keyword(s):  
Metal Binding ◽  
Binding Properties
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