Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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A modified dinucleotide for site-specific RNA-labelling by transcription priming and click chemistry

Chemical Communications ◽

10.1039/c3cc46132g ◽

2014 ◽

Vol 50 (11) ◽

pp. 1313-1316 ◽

Cited By ~ 29

Author(s):

Ayan Samanta ◽

André Krause ◽

Andres Jäschke

Keyword(s):

Click Chemistry ◽

Site Specific

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Site-Specific Surface Functionalization via Microchannel Cantilever Spotting (µCS): Comparison between Azide-Alkyne and Thiol-Alkyne Click Chemistry Reactions

Small ◽

10.1002/smll.201800131 ◽

2018 ◽

Vol 14 (21) ◽

pp. 1800131 ◽

Cited By ~ 9

Author(s):

Seyed Mohammad Mahdi Dadfar ◽

Sylwia Sekula-Neuner ◽

Uwe Bog ◽

Vanessa Trouillet ◽

Michael Hirtz

Keyword(s):

Specific Surface ◽

Click Chemistry ◽

Surface Functionalization ◽

Site Specific

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Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3757-3765.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3757-3765

Author(s):

J W Chen ◽

B R Evans ◽

S H Yang ◽

H Araki ◽

Y Oshima ◽

...

Keyword(s):

Active Site ◽

Dna Cleavage ◽

Substrate Binding ◽

Point Mutations ◽

Transfer Reactions ◽

Strand Transfer ◽

Site Specific ◽

Arginine Residues ◽

Active Site Tyrosine ◽

Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

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BIS-ANS Binding To Human Thrombins

10.1055/s-0038-1652085 ◽

1981 ◽

Author(s):

G Metz ◽

L J Berliner

Keyword(s):

Active Site ◽

Unique Feature ◽

Enhanced Fluorescence ◽

Ans Binding ◽

Site Specific ◽

Catalytic Center ◽

Strong Binding

Bis-anilinonaphthalenesulfonate(bis-ANS) binds extremely strongly to the human thrombins accompanied by a much enhanced fluorescence for the dye. One unique feature of bis-ANS binding is an enhancement of TAME hydrolysis by α-thrombin to approximately 175% times the rate for native α-thrombin at pH 8.1 (0 M NaCl). Non-coagulant γ-thrombin is distinguished from highly coagulant α-thrombin by enhancing bis-ANS fluorescence ∼2 times more than that by the latter form. The very strong binding of this dye Kdiss<10-8 M, pH 6.5, 0.05 M Phosphate, 0.75 M NaCl) as welras its apparent binding loci situated near, but not obstructing, the catalytic center displays the powerfully useful properties of this probe, extremely sensitive to thrombin environment while not inhibiting its action. This is to be compared with the active site specific fluorophore inhibitor DAPA which prevents binding of other ligands to the thrombin active site.

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