Identification and Purification of a Non-Ceruloplasmin Ferroxidase of Human Serum

Topham, Richard W.; Frieden, Earl

doi:10.1016/s0021-9258(18)62590-6

Cited by 60 publications

(13 citation statements)

References 18 publications

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“…However, despite all activity being ablated when using the ferric gain assay (Figure Bi), a small proportion of retentate activity was not inhibited by azide when measured by TF loading (1.44 ± 0.13 μM Fe 3+ /min) (Figure Bii). This is consistent with previous TF loading reports identifying the presence of a non-CP ferroxidase activity in serum. , End point quantitation of Fe 2+ loss corroborated the production of Fe 3+ during the assays in both the presence and absence of TF (Figure C).…”

supporting

confidence: 91%

“…This is consistent with previous TF loading reports identifying the presence of a non-CP ferroxidase activity in serum. 11,17 End point quantitation of Fe 2+ loss corroborated the production of Fe 3+ during the assays in both the presence and absence of TF (Figure 1C).…”

supporting

confidence: 63%

“…7 Decreased CP oxidase activity has been reported in serum from patients suffering from Alzheimer's disease (AD) 9 and PD. 10 The incorporation of iron into TF has been used to measure ferroxidase activity indirectly, 4,11 but two main controversies on its accuracy arise from a reliance on nonphysiological (low) pH in order to suppress auto-oxidation, as well as the requirement for indirect colorimetric detection of holo-TF formation, which is a rate-limiting extraneous step in appraising the enzyme kinetics. 1 A more direct measurement of ferroxidase activity is to assay Fe 2+ loss 12,13 and Fe 3+ gain 12,14 but buffer conditions are not physiological in these systems, and TF is absent.…”

mentioning

confidence: 99%

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Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer’s Disease Subjects

Lam

Wong

Frugier

et al. 2017

ACS Chem. Neurosci.

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Ferroxidase activity has been reported to be altered in various biological fluids in neurodegenerative disease, but the sources contributing to the altered activity are uncertain. Here we assay fractions of serum and cerebrospinal fluid with a newlyvalidated triplex ferroxidase assay. Our data indicate that while ceruloplasmin, a multicopper ferroxidase, is the predominant source of serum activity, activity in CSF predominantly derives from a <10kDa component, specifically from polyanions such as citrate and phosphate. We confirm that in human biological samples, ceruloplasmin activity in serum is decreased in AlzheimerÕs disease, but in CSF a reduction of activity in AlzheimerÕs disease originates from the polyanion component. Multicopper ferroxidase inhibition with sodium azide 12 verified that CP was the major component of retentate activity (Fig. 1B). However, despite all activity being ablated when using the Ferric Gain assay (Fig. 1Bi), a small proportion of retentate activity Keywordswas not inhibited by azide when measured by TF loading (1.44 ± 0.13 µM Fe 3+ /min) ( Fig. 1Bii). This is consistent with previous TF loading reports identifying the presence of a non-CP ferroxidase activity in serum 11,17 . End point quantitation of Fe 2+ loss corroborated the production of Fe 3+ during the assays in both the presence and absence of TF (Fig. 1C).Triplex assay parameters for CSF were found to be as previously determined for purified CP 1 and human serum ( Fig. 1), at an optimal CSF volume of 20 µl (Fig. S2B). In contrast to serum fractions ( Fig. 1 In assaying the same volume (5µl) of serum and CSF in both the ÔFerric GainÕ and ÔTF loadingÕ components of the triplex assay, activity was markedly greater in serum (Fig. S3A). Upon fractionation of either biological fluid, retentate had markedly greater activity from serum ( Fig. S3B) and negligible activity from CSF ( Fig. 2A).Despite this greater percentage of filtrate activity in total CSF, when compared to the same serum volume this fractionÕs activity only had a minor elevation in Ferric Gain Azide (2.5mM) did not inhibit CSF activity (Fig. 2B), excluding the source of the activity being a multicopper ferroxidase e.g. CP. The absence of CP activity in human CSF is in alignment with normal levels of CP (~1.5 µg/ml) 18 being <1% of those ! 6 found in serum (200-500µg/ml) 19 . Consistent with the previously reported level of CP in CSF, recombinant CP at this concentration (~2.5 µg/ml) is outside the active range of our assay (<20nM; Fig. S4). Our findings are similar to the activity that has been reported to arise from <10kDa species in CSF from healthy and sporadic CreutzfeldtJakob disease (sCJD) patients 14 , but at variance with a report that ferroxidase activity in CSF originates from CP 13 .To identify whether the ferroxidase activity from the filtrate was of protein enzyme origin, fractionated serum and CSF were treated with proteinase K. In serum, the protease abolished the retentate activity, but had no effect on the filtrate activity ( Fig. 3A...

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supporting

confidence: 91%

supporting

confidence: 63%

mentioning

confidence: 99%

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Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer’s Disease Subjects

Lam

Wong

Frugier

et al. 2017

ACS Chem. Neurosci.

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“…The purification and partial characterization of a nonceruloplasmin ferroxidase (ferroxidase II) from whole human sera and the Cohn IV-1 fraction of human plasma have been previously reported (Topham and Frieden, 1970;Sung and Topham, 1973;Topham and Johnson, 1974;Topham et al, 1975). Kinetic studies (Topham and Johnson, 1974) have shown that ferroxidase II would be capable of functioning as an alternative for ferroxidase I (ceruloplasmin) in human serum and as the major ferroxidase in sera in species that contain low ferroxidase I levels.…”

mentioning

confidence: 98%

“…Experimental Procedure Materials Ferroxidase 11. Highly purified ferroxidase II was obtained by a slight modification of the method previously described (Topham and Frieden, 1970). It was necessary to pass partially purified ferroxidase II over an Agarose A-50m column two successive times rather than once to obtain a preparation which f From the Department of Chemistry, University of Richmond, Richmond, Virginia 23173.…”

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confidence: 99%

Dissociation and reconstitution of human ferroxidase II

Lykins¹,

Akey²,

Christian³

et al. 1977

Biochemistry

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The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.

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Ceruloplasmin: The Copper Transport Protein With Essential Oxidase Activity

Frieden¹,

Hs²

1976

Advances in Enzymology - And Related Areas of Molecular Biology

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Identification and Purification of a Non-Ceruloplasmin Ferroxidase of Human Serum

Cited by 60 publications

References 18 publications

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer’s Disease Subjects

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer’s Disease Subjects

Dissociation and reconstitution of human ferroxidase II

Ceruloplasmin: The Copper Transport Protein With Essential Oxidase Activity

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