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The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma.

Takahashi K, Kondo T, Yoshikawa Y, Watanabe K, Orino K - Acta Vet. Scand. (2013)

Bottom Line: There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated.Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan. orino@vmas.kitasato-u.ac.jp.

ABSTRACT

Background: Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings: Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions: Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

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Inhibition of the binding between holoferritin and purified fibrinogen or fibrinogen in heat-treated plasma by hemin, metal free-, Sn-, or Zn-PPIX. Aliquots (100 μL) of 10 μg/mL of purified horse fibrinogen (A) or heated-horse plasma sample (B) as described in “Figure 1” were added to wells of holo-ferritin-coated plate (1 pmol/well) and hemin (solid bar), Sn- (gray bar), Zn- (dotted bar), or metal free-PPIX (open bar) was added to a final concentration of 10 μmol/L. Fibrinogen bound to the wells was detected as already described in “Figure 1”. Binding activity (%) was determined by comparison to the control (100%) in the absence of each inhibitor. a: P < 0.01 compared to the binding activity in the presence of hemin; b: P < 0.01 compared to the binding activity in the presence of Sn-PPIX; c: P < 0.01 compared to the binding activity in the presence of Zn-PPIX. Each data represents mean ± SD of four determinations.
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Figure 2: Inhibition of the binding between holoferritin and purified fibrinogen or fibrinogen in heat-treated plasma by hemin, metal free-, Sn-, or Zn-PPIX. Aliquots (100 μL) of 10 μg/mL of purified horse fibrinogen (A) or heated-horse plasma sample (B) as described in “Figure 1” were added to wells of holo-ferritin-coated plate (1 pmol/well) and hemin (solid bar), Sn- (gray bar), Zn- (dotted bar), or metal free-PPIX (open bar) was added to a final concentration of 10 μmol/L. Fibrinogen bound to the wells was detected as already described in “Figure 1”. Binding activity (%) was determined by comparison to the control (100%) in the absence of each inhibitor. a: P < 0.01 compared to the binding activity in the presence of hemin; b: P < 0.01 compared to the binding activity in the presence of Sn-PPIX; c: P < 0.01 compared to the binding activity in the presence of Zn-PPIX. Each data represents mean ± SD of four determinations.

Mentions: The binding mechanism of plasma in horse plasma and ferritin has not been revealed due to non-binding of fibrinogen to ferritin. Subjecting horse plasma to heat treatment at 60°C for 30 min resulted in maximum binding of plasma fibrinogen to ferritin-coated microwells (Additional file 1: Data S1). Fibrinogen in heated-treated plasmas showed significant higher binding activity with holoferritin than with apoferritin as in purified horse fibrinogen (Figure 1A and B). Human fibrinogen showed heat stability (68°C, 10 h) in the pasteurization process [12]. However, heat denaturation of fibrinogen occurred by divalent cations such as Ca2+ (2 mmol/L) and Zn2+ (20 μmol/L) [13]. Therefore, a dilution (100-fold) of plasma may eliminate the possibility of fibrinogen denaturation by heat-treatment and divalent cations [14]. Therefore, we suggested that binding between fibrinogen and ferritin is heme-mediated as in apolipoprotein B and α-casein because reducing treatment of holoferritin releases heme as well as iron [3,10]. Binding between purified fibrinogen or plasma fibrinogen to holoferritin was significantly inhibited by hemin and Sn-PPIX, but not by Zn-PPIX or metal free-PPIX (Figure 2A and B) as calculated the binding activity (%) as 100% for the control in the absence of each inhibitor. This result agrees with the observation that Sn-PPIX is the most potent competitive inhibitor of heme oxygenase reacting with heme as substrate [15]. Binding between purified fibrinogen or plasma fibrinogen to holoferritin was not blocked by ferrous ammonium sulfate (Fe2+) or ammonium iron sulfate (Fe3+), even at a concentration of 1 mmol/L (data not shown), suggesting that the binding of fibrinogen to ferritin is not iron-dependent or not necessary for only iron. These results demonstrated that horse fibrinogen strongly recognizes iron or tin complexed with the heme- or the hemin-ring. Whereas the potency of the inhibitors used to block binding between purified fibrinogen and holoferritin was in the order hemin > Sn-PPIX > Zn-PPIX, Sn-PPIX was the most potent inhibitor when using heat-treated plasma, and Zn-PPIX did not show any inhibition. Additionally, PPIX enhanced the binding between plasma fibrinogen and holoferritin different from purified fibrinogen. The different inhibitory effects of various PPIX derivatives on the ferritin-binding in purified fibrinogen and plasma fibrinogen remains to be elucidated. Plasma contains factors such as hemopexin as a heme-binding protein [16] and albumin and α2-macroglobulin that bind Zn ion [17], suggesting that these interactions result in apparent lower inhibitor concentrations compared to purified fibrinogen. Plasma may contain some factors that interact with PPIX to enhance binding activity between fibrinogen and ferritin.


The presence of heat-labile factors interfering with binding analysis of fibrinogen with ferritin in horse plasma.

Takahashi K, Kondo T, Yoshikawa Y, Watanabe K, Orino K - Acta Vet. Scand. (2013)

Inhibition of the binding between holoferritin and purified fibrinogen or fibrinogen in heat-treated plasma by hemin, metal free-, Sn-, or Zn-PPIX. Aliquots (100 μL) of 10 μg/mL of purified horse fibrinogen (A) or heated-horse plasma sample (B) as described in “Figure 1” were added to wells of holo-ferritin-coated plate (1 pmol/well) and hemin (solid bar), Sn- (gray bar), Zn- (dotted bar), or metal free-PPIX (open bar) was added to a final concentration of 10 μmol/L. Fibrinogen bound to the wells was detected as already described in “Figure 1”. Binding activity (%) was determined by comparison to the control (100%) in the absence of each inhibitor. a: P < 0.01 compared to the binding activity in the presence of hemin; b: P < 0.01 compared to the binding activity in the presence of Sn-PPIX; c: P < 0.01 compared to the binding activity in the presence of Zn-PPIX. Each data represents mean ± SD of four determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016576&req=5

Figure 2: Inhibition of the binding between holoferritin and purified fibrinogen or fibrinogen in heat-treated plasma by hemin, metal free-, Sn-, or Zn-PPIX. Aliquots (100 μL) of 10 μg/mL of purified horse fibrinogen (A) or heated-horse plasma sample (B) as described in “Figure 1” were added to wells of holo-ferritin-coated plate (1 pmol/well) and hemin (solid bar), Sn- (gray bar), Zn- (dotted bar), or metal free-PPIX (open bar) was added to a final concentration of 10 μmol/L. Fibrinogen bound to the wells was detected as already described in “Figure 1”. Binding activity (%) was determined by comparison to the control (100%) in the absence of each inhibitor. a: P < 0.01 compared to the binding activity in the presence of hemin; b: P < 0.01 compared to the binding activity in the presence of Sn-PPIX; c: P < 0.01 compared to the binding activity in the presence of Zn-PPIX. Each data represents mean ± SD of four determinations.
Mentions: The binding mechanism of plasma in horse plasma and ferritin has not been revealed due to non-binding of fibrinogen to ferritin. Subjecting horse plasma to heat treatment at 60°C for 30 min resulted in maximum binding of plasma fibrinogen to ferritin-coated microwells (Additional file 1: Data S1). Fibrinogen in heated-treated plasmas showed significant higher binding activity with holoferritin than with apoferritin as in purified horse fibrinogen (Figure 1A and B). Human fibrinogen showed heat stability (68°C, 10 h) in the pasteurization process [12]. However, heat denaturation of fibrinogen occurred by divalent cations such as Ca2+ (2 mmol/L) and Zn2+ (20 μmol/L) [13]. Therefore, a dilution (100-fold) of plasma may eliminate the possibility of fibrinogen denaturation by heat-treatment and divalent cations [14]. Therefore, we suggested that binding between fibrinogen and ferritin is heme-mediated as in apolipoprotein B and α-casein because reducing treatment of holoferritin releases heme as well as iron [3,10]. Binding between purified fibrinogen or plasma fibrinogen to holoferritin was significantly inhibited by hemin and Sn-PPIX, but not by Zn-PPIX or metal free-PPIX (Figure 2A and B) as calculated the binding activity (%) as 100% for the control in the absence of each inhibitor. This result agrees with the observation that Sn-PPIX is the most potent competitive inhibitor of heme oxygenase reacting with heme as substrate [15]. Binding between purified fibrinogen or plasma fibrinogen to holoferritin was not blocked by ferrous ammonium sulfate (Fe2+) or ammonium iron sulfate (Fe3+), even at a concentration of 1 mmol/L (data not shown), suggesting that the binding of fibrinogen to ferritin is not iron-dependent or not necessary for only iron. These results demonstrated that horse fibrinogen strongly recognizes iron or tin complexed with the heme- or the hemin-ring. Whereas the potency of the inhibitors used to block binding between purified fibrinogen and holoferritin was in the order hemin > Sn-PPIX > Zn-PPIX, Sn-PPIX was the most potent inhibitor when using heat-treated plasma, and Zn-PPIX did not show any inhibition. Additionally, PPIX enhanced the binding between plasma fibrinogen and holoferritin different from purified fibrinogen. The different inhibitory effects of various PPIX derivatives on the ferritin-binding in purified fibrinogen and plasma fibrinogen remains to be elucidated. Plasma contains factors such as hemopexin as a heme-binding protein [16] and albumin and α2-macroglobulin that bind Zn ion [17], suggesting that these interactions result in apparent lower inhibitor concentrations compared to purified fibrinogen. Plasma may contain some factors that interact with PPIX to enhance binding activity between fibrinogen and ferritin.

Bottom Line: There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated.Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan. orino@vmas.kitasato-u.ac.jp.

ABSTRACT

Background: Horse fibrinogen has been identified as a plasma specific ferritin-binding protein. There are two ways in the binding of ferritin-binding protein with ferritin: one is direct binding and the other is indirect binding which is heme-mediated. The aim of this study was to analyze the binding between horse fibrinogen and ferritin.

Findings: Although fibrinogen in horse plasma did not show the binding to ferritin coated on the plate wells, after following heat-treatment (60°C, 30 min) of horse plasma, plasma fibrinogen as well as purified horse fibrinogen bound to plates coated with horse spleen ferritin, but not with its apoferritin which lost heme as well as iron after the treatment of reducing reagent. Binding of purified or plasma fibrinogen to ferritin was inhibited by hemin and Sn-protoporphyrin IX (Sn-PPIX), but not by PPIX or Zn-PPIX.

Conclusions: Heat-treatment of horse plasma enabled plasma fibrinogen to bind to plate well coated with holo-ferritin. From the binding analysis of fibrinogen and ferritin, it is suggested that horse fibrinogen recognized iron or tin in complexed with the heme- or the hemin-ring, and also suggest that some fibrinogens circulate in the form of a complex with ferritin and/or heat-labile factors which inhibit the binding of fibrinogen with ferritin.

Show MeSH
Related in: MedlinePlus