Temperature-dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation.
ADAMTS13 Protein
/ antagonists & inhibitors
Calcium
/ metabolism
Catalytic Domain
/ drug effects
Chelating Agents
/ pharmacology
Citric Acid
/ pharmacology
Dynamic Light Scattering
Enzyme Stability
/ drug effects
Fluorescence Resonance Energy Transfer
Humans
In Vitro Techniques
Mass Spectrometry
Protein Conformation
/ drug effects
Purpura, Thrombotic Thrombocytopenic
/ blood
Recombinant Proteins
/ chemistry
Spectroscopy, Fourier Transform Infrared
Temperature
Zinc
/ metabolism
ADAMTS13 protein
blood plasma
protein conformation
protein stability
zinc
Journal
Journal of thrombosis and haemostasis : JTH
ISSN: 1538-7836
Titre abrégé: J Thromb Haemost
Pays: England
ID NLM: 101170508
Informations de publication
Date de publication:
06 2019
06 2019
Historique:
received:
23
10
2018
accepted:
20
03
2019
pubmed:
23
4
2019
medline:
1
7
2020
entrez:
23
4
2019
Statut:
ppublish
Résumé
The catalytic domain of ADAMTS13 possesses one Zn To address the stability of ADAMTS13 in citrated human plasma. ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering. ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca Zn
Sections du résumé
BACKGROUND
The catalytic domain of ADAMTS13 possesses one Zn
OBJECTIVES
To address the stability of ADAMTS13 in citrated human plasma.
METHODS
ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering.
RESULTS
ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca
CONCLUSIONS
Zn
Identifiants
pubmed: 31006963
doi: 10.1111/jth.14440
pmc: PMC6850365
pii: S1538-7836(22)04440-3
doi:
Substances chimiques
Chelating Agents
0
Recombinant Proteins
0
Citric Acid
2968PHW8QP
ADAMTS13 Protein
EC 3.4.24.87
ADAMTS13 protein, human
EC 3.4.24.87
Zinc
J41CSQ7QDS
Calcium
SY7Q814VUP
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
995-1002Informations de copyright
© 2019 Shire International GmbH. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.
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