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 language | English |
|---|---|
| Pages (from-to) | 183-190 |
| Number of pages | 8 |
| Journal | Clinical Chemistry and Enzymology Communications |
| Volume | 3 |
| Issue number | 2-3 |
| Publication status | Published - 1990 |
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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 journal › Article
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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.
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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 -