Fluorogenic substrate for ADAMTS13
Disclosed are fluorogenic substrates for measuring ADAMTS13 activity or ADAMTS13 inhibitor activity. Substrates can comprise an oligopeptide which can consist of up to 80 amino acids of sequence of von Willebrand Factor (VWF). The oligopeptide can include modifications of sequence of VWF, including an amino-terminal glycine, a scissile Y-M peptide, and a cysteine substitution located from 1 to 12 amino acids from the scissile Y-M in the carboxy terminal direction. A substrate can further comprise a fluorophore and a fluorescence quencher bound to the oligopeptide on opposite sides of the scissile Y-M peptide, wherein the fluorescence quencher is not identical to the fluorophore. An oligopeptide can be encoded by a nucleic acid sequence which can also encode a His tag. An oligopeptide can be expressed in a cell or microorganism. Also disclosed are methods of using a fluorogenic substrate to measure ADAMTS13 activity or ADAMTS13 inhibitor activity.
1. A probe comprising:
an oligopeptide consisting of no more than 80 amino acids of sequence of von Willebrand Factor (VWF), said oligopeptide comprising a scissile Y1605-M1606 peptide of a VWF sequence set forth as SEQ ID NO: 3, a cysteine substitution located from 1 to 12 amino acids from the scissile Y-M in the carboxy terminal direction, and a C-terminal segment;
a fluorophore: and
a fluorescence quencher,
wherein the fluorophore and the fluorescence quencher are bound to the oligopeptide on opposite sides of the scissile Y-M peptide and wherein the oligopeptide comprises a substitution of lysine K1617 of a VWF sequence set forth as SEQ ID NO: 3 with an amino acid that does not comprise a primary amine on its side chain.
2. A probe in accordance with claim 1 , wherein the cysteine substitution is a substitution of an amino acid located at least 3 amino acids from the scissile Y-M peptide.
3. A probe in accordance with claim 1 , wherein the cysteine substitution is a N1610C substitution of a VWF sequence set forth as SEQ ID NO: 3.
4. A probe in accordance with claim 1 wherein the oligopeptide consists of the sequence set forth as SEQ ID NO: 2.
5. A probe in accordance with claim 1 . wherein the fluorophore has an absorption maximum >550 nm.
6. A probe in accordance with claim 1 , wherein the fluorophore has an emission maximum >600 nm.
7. A probe in accordance with claim 1 wherein the fluorophore is selected from the group consisting of Alexa Fluor®594 maleimide (fluorophore)
and DyLight®633 maleimide (fluorophore)
8. A probe in accordance with claim 1 wherein the quencher has an absorption maximum >550 nm.
9. A probe in accordance with claim 1 , wherein at least one of the fluorophore and the quencher comprises at least one sulfate.
10. A probe in accordance with claim 1 , wherein the quencher is selected from the group consisting of QSY®(fluorescence quencher) 21- succinimidyl ester and IRDye® (infrared dye) QC-1 N-hydroxy succinimidyl ester.
11. A probe in accordance with claim 1 , wherein the probe is soluble in water at >50 μM.
12. A method of determining presence, absence or quantity of ADAMTS13 activity in a sample, comprising;
forming a mixture comprising a sample and a probe of claim 1 ; and
measuring fluorescence at one or more time points after forming the mixture, wherein the sample is diluted less than 20-fold.
13. A method in accordance with claim 12 , wherein the sample is selected from the group consisting of a serum sample, an undiluted serum sample, a concentrated serum sample, a plasma sample, an undiluted plasma sample and a concentrated plasma sample.
14. A method of determining presence, absence or quantity of ADAMTS13 inhibitor activity in a sample, comprising;
forming a mixture comprising a sample, as source of ADAMTS13, and a probe of claim 1 ; and
measuring fluorescence at one or more time points after forming the mixture.
15. A method in accordance with claim 14 , further comprising inactivating ADAMTS13 activity endogenous to the sample prior to forming the mixture.
16. A method in accordance with claim 14 , wherein the source of ADAMTS13 is selected from the group consisting of normal plasma, recombinant ADAMTS13 and a combination thereof.
17. A method in accordance with claim 14 , wherein the sample is selected from the group consisting of a serum sample, an undiluted serum sample, a concentrated serum sample, a plasma sample, an undiluted plasma sample and a concentrated plasma sample.
18. A probe in accordance with claim 1 , wherein the C-terminal segment consists of sequence EAPDLVLQR(SEQ ID NO: 6).
19. A probe comprising:
an oligopeptide consisting of no more than 80 amino acids of sequence of von Willebrand Factor (VWF), said oligopeptide comprising a scissile Y-M peptide, a cysteine substitution located from 1 to 12 amino acids from the scissile Y-M in the carboxy terminal direction, and a C-terminal segment;
as fluorophore; and
as fluorescence quencher,
wherein the fluorophore and the fluorescence quencher are bound to the oligopeptide on opposite sides of the scissile Y-M peptide and wherein the oligopeptide consists of the sequence set forth as SEQ ID NO: 2.