Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening

2021 ◽  
Author(s):  
Hong-Ming Chen ◽  
Seyed Amirhossein Nasseri ◽  
Peter Rahfeld ◽  
Jacob F. Wardman ◽  
Maurits Kohsiek ◽  
...  
Keyword(s):  
Fluorogenic Substrates

Synthesis of sensitive coumarin α-GalNAc glycosides as substrates for droplet-based screening of GalNAcases.

Ultrasensitive Fluorogenic Substrates for Serine Proteases

1997 ◽  
Vol 78 (04) ◽  
pp. 1193-1201 ◽  
Author(s):  
Saulius Butenas ◽  
Maria E DiLorenzo ◽  
Kenneth G Mann
Keyword(s):  
Serine Proteases ◽  
Factor Viia ◽  
Activated Protein C ◽  
Factor Xa ◽  
High Specificity ◽  
High Rate ◽  
Fluorogenic Substrates ◽  
Type Plasminogen Activator ◽  
Factor Xia ◽  
Coagulation And Fibrinolysis

SummarySelective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a kcat value of 170 s-1 and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by > 10,000-fold and > 100-fold for activated protein C (APC). Seven of these substrates have a over 100 s-1 and three of them have a KM below 1 μM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor Vila/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.

New fluorogenic substrates for renin

1981 ◽  
Vol 110 (1) ◽  
pp. 232-239 ◽  
Author(s):  
Kazuo Murakami ◽  
Tamiko Ohsawa ◽  
Shigehisa Hirose ◽  
Katsumi Takada ◽  
Shumpei Sakakibara
Keyword(s):  
Fluorogenic Substrates

Selective Neurotensin-Derived Internally Quenched Fluorogenic Substrates for Neurolysin (EC 3.4.24.16): Comparison with Thimet Oligopeptidase (EC 3.4.24.15) and Neprilysin (EC 3.4.24.11)

2001 ◽  
Vol 292 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Vitor Oliveira ◽  
Marcelo Campos ◽  
Jefferson P. Hemerly ◽  
Emer S. Ferro ◽  
Antonio C.M. Camargo ◽  
...  
Keyword(s):  
Fluorogenic Substrates ◽  
Thimet Oligopeptidase

An alternative approach for the synthesis of fluorogenic substrates of endo-β-(1→4)-xylanases and some applications

2008 ◽  
Vol 343 (3) ◽  
pp. 541-548 ◽  
Author(s):  
Mária Vršanská ◽  
Wim Nerinckx ◽  
Marc Claeyssens ◽  
Peter Biely
Keyword(s):  
Fluorogenic Substrates ◽  
Alternative Approach

Detecting enzymes in living cells using fluorogenic substrates

1993 ◽  
Vol 3 (3) ◽  
pp. 119-127 ◽  
Author(s):  
Richard P. Haugland ◽  
Iain D. Johnson
Keyword(s):  
Living Cells ◽  
Fluorogenic Substrates

4-Methylumbelliferyl esters as fluorogenic substrates for proteases

1983 ◽  
Vol 5 (2) ◽  
pp. 137-142 ◽  
Author(s):  
C.J. Gray ◽  
C.J.S.J. D'Silva ◽  
J. Boukouvalas ◽  
S.A. Barker
Keyword(s):  
Fluorogenic Substrates

Evaluation of fluorogenic substrates in the assessment of digestive enzymes in a decapod crustacean Maja brachydactyla larvae

Aquaculture ◽  
2008 ◽  
Vol 282 (1-4) ◽  
pp. 90-96 ◽  
Author(s):  
Guiomar Rotllant ◽  
Francisco Javier Moyano ◽  
Mireia Andrés ◽  
Manuel Díaz ◽  
Alicia Estévez ◽  
...  
Keyword(s):  
Digestive Enzymes ◽  
Decapod Crustacean ◽  
Fluorogenic Substrates

scholarly journals Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates.

2001 ◽  
Vol 48 (4) ◽  
pp. 995-1002 ◽  
Author(s):  
M Szabelski ◽  
K Stachowiak ◽  
W Wiczk
Keyword(s):  
Incubation Time ◽  
Active Sites ◽  
Initial Rate ◽  
Substrate Binding ◽  
Rapid Decrease ◽  
Fluorogenic Substrates ◽  
Binding Process ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).

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