Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase

P. Ascenzi, G. Amiconi, E. Menegatti, M. Guarneri

Research output: Contribution to journalArticle

Abstract

A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu1, Lys77-, Val442-, and Val561-plasmin, as well as of active human Glu1-, Lys77-, Val442- and Val561-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu1-, Lys77-, Val442- and Val561-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu1-, Lys77-, Val442- and Val561-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu1-, Lys77- Val442- and Val561-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10-3 μM.

Original languageEnglish
Pages (from-to)183-190
Number of pages8
JournalClinical Chemistry and Enzymology Communications
Volume3
Issue number2-3
Publication statusPublished - 1990

Fingerprint

Fibrinolysin
Serine Proteases
Thrombin
Hydrolysis
Aprotinin
Hirudins
Enzymes
Lysine
Hirudo medicinalis
Assays
Esters
Leeches
Kinetics
Substrates
Chemical analysis

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase. / Ascenzi, P.; Amiconi, G.; Menegatti, E.; Guarneri, M.

In: Clinical Chemistry and Enzymology Communications, Vol. 3, No. 2-3, 1990, p. 183-190.

Research output: Contribution to journalArticle

@article{88ae957808a549e48625a4cc3fc16b44,
title = "Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase",
abstract = "A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu1, Lys77-, Val442-, and Val561-plasmin, as well as of active human Glu1-, Lys77-, Val442- and Val561-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu1-, Lys77-, Val442- and Val561-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu1-, Lys77-, Val442- and Val561-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu1-, Lys77- Val442- and Val561-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10-3 μM.",
author = "P. Ascenzi and G. Amiconi and E. Menegatti and M. Guarneri",
year = "1990",
language = "English",
volume = "3",
pages = "183--190",
journal = "Clinical Chemistry and Enzymology Communications",
issn = "0892-2187",
publisher = "Harwood Academic Publishers",
number = "2-3",

}

TY - JOUR

T1 - Determination of active human α-, β- and γ-thrombin and human Glu1-, Lys77, Val442- and Val561-plasmin concentration in the presence of cognate serine proteinase

AU - Ascenzi, P.

AU - Amiconi, G.

AU - Menegatti, E.

AU - Guarneri, M.

PY - 1990

Y1 - 1990

N2 - A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu1, Lys77-, Val442-, and Val561-plasmin, as well as of active human Glu1-, Lys77-, Val442- and Val561-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu1-, Lys77-, Val442- and Val561-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu1-, Lys77-, Val442- and Val561-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu1-, Lys77- Val442- and Val561-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10-3 μM.

AB - A spectrophotometric method for the determination of active human α-, β- and γ-thrombin concentration, in the presence of human Glu1, Lys77-, Val442-, and Val561-plasmin, as well as of active human Glu1-, Lys77-, Val442- and Val561-plasmin concentration, in the presence of human α-, β- and γ-thrombin, is reported. This assay method has been developed from the quantitative analysis of kinetics for the hydrolysis of the N-α-carbobenzoxy-L-lysine p-nitrophenyl ester catalyzed by human α-, β- and γ-thrombin, in the presence of human Glu1-, Lys77-, Val442- and Val561-plasmin selectively inhibited by the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor), and by human Glu1-, Lys77-, Val442- and Val561-plasmin, in the presence of human α-, β- and γ-thrombin selectively inhibited by hirudin, the thrombin-specific inhibitor from leech Hirudo medicinalis. The active enzyme concentration has been estimated, under conditions where [S](o) >> K(m), from the dependence of the initial rate for substrate hydrolysis (i.e., V) on the enzyme concentration (i.e., [E](o)) taking into account the proportionality constant k(cat) (i.e., V = k(cat) · [E](o)). The minimum active human α-, β- and γ-thrombin and human Glu1-, Lys77- Val442- and Val561-plasmin concentration which may be evaluated from the analysis of the experimental data corresponds to 2.5 x 10-3 μM.

UR - http://www.scopus.com/inward/record.url?scp=0025025999&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025025999&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:0025025999

VL - 3

SP - 183

EP - 190

JO - Clinical Chemistry and Enzymology Communications

JF - Clinical Chemistry and Enzymology Communications

SN - 0892-2187

IS - 2-3

ER -