Determination of Plasminogen by Means of a Chroniogenic Peptide Substrate

1975 ◽  
Author(s):  
P. Friberger ◽  
G. Axelsson ◽  
K. Korsan-Bengtsen
Keyword(s):  
Ionic Strength ◽  
Linear Relationship ◽  
Reaction Rate ◽  
High Rate ◽  
Chromogenic Substrate ◽  
Peptide Substrate ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Slight Inhibition

Plasmin splits the chromogenic substrate B2-Phe-Val-Arg-pNA (S-2160, Bofors) at a relatively high rate. Standard plasmin in glycerol obtained from Nat. Inst, for Biol. Stand, and Contr., London, was tested in a system with Tris buffer of varying pH and ionic strength. The pH optimum for the reaction was found to be 7.4. Variations in ionic strength between 0.05–0.1 had insignificant influence but at higher ionic strength there was a slight inhibition. A linear relationship was found between plasmin and AOD/min. At optimum pH and a final substrate concentration of 0.2 mM 0.1 CTA unit corresponds to approximately 0.10 nkat. Purified plasminogen (AB Kabi, Stockholm, Sweden) in the concentrations 0.02–0.2 CU/ml was activated optimally with streptokinase (Kabikinase® ) in the concentrations 500–2000 IU/ml. Higher concentration gave inhibition. The activity of streptokinase activated plasminogen increased with a decreasing ionic strength. A linear relationship was found between streptokinase activated plasminogen and AOD/min. Approximately 3,000 Plong/units per ml of urokinase was needed to obtain the same activation as with optimal streptokinase concentration. The method has been used for determination of plasminogen in plasma. With final dilution of plasma in the range 1/20–1/200 activated by streptokinase (2000 IU/ml) in a system of pH 8.2, I = 0.05, a linear relationship was found between plasma dilution and AOD/min. The reproducibility in a series of tests is good (variation coefficient < 3%) and with insignificant interference by inhibitors. The determinations were easily carried out in a simple spectrometer (405 nm) and in an automatic reaction rate analyzer (LKB 8600, 410 nm).

scholarly journals Immobilization of Isolated Lipase From Moldy Copra(Aspergillus Oryzae)

2011 ◽  
Vol 8 (2) ◽  
pp. 896-902
Author(s):  
Seniwati Dali ◽  
A. B. D. Rauf Patong ◽  
M. Noor Jalaluddin ◽  
Pirman ◽  
Baharuddin Hamzah
Keyword(s):  
Thermal Stability ◽  
Aspergillus Oryzae ◽  
Immobilized Lipase ◽  
Optimum Temperature ◽  
Adsorption Method ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Recovery Technique ◽  
Free Lipase

Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase fromAspergillus oryzae. Lipase was partially purified from the culture supernatant ofAspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum temperature, thermal stability and reusability were carried out. The results showed that free lipase had optimum pH 8,2 and optimum temperature 35 °C while the immobilized lipase had optimum 8,2 and optimum temperature 45 °C. The thermal stability of the immobilized lipase, relative to that of the free lipase, was markedly increased. The immobilized lipase can be reused for at least six times.

Levels of Platelet Factor 3 in Citrate Plasma and in Whole Blood as Determined by A Chromogenic Peptide Substrate Assay

1979 ◽  
Author(s):  
H. Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PCE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PCE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PCE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PCE 1/theophy11ine anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and βtg are released in parallel.

Studies on Nature of So-Called Antiplasmin Measured By Chromogenic Substrate S-2251 and Its Automatic Determination With ABA 100

1979 ◽  
Author(s):  
M Kato ◽  
M Fujimaki ◽  
K Fukutake ◽  
A Maki ◽  
M Hibino
Keyword(s):  
Protease Inhibitors ◽  
Reaction Rate ◽  
Chromogenic Substrate ◽  
Excellent Correlation ◽  
Automatic Determination ◽  
Substrate Solution ◽  
Simultaneous Addition ◽  
Single Radial Immunodiffusion ◽  
At Iii

Using chromogenic substrate S-2251, authors studied on the nature of so-called antiplasmin and on the application of Coatest to automatic determination. As to several protease inhibitors, correlation performed vs single radial immunodiffusion yielded a regression of Y=19.5X-18.6 and r=0.91 for α2PI ;r=-0.15, 0.13, 0.46 0.28 for α2M, α2AT, AT-III and CIINA respectively. Purified protease inhibitors except α2PI did not exhibit immediate inhibition to human plasmin. The automation of chromogenic substrate method was studied with ABA Bichromatic Analyser Model 100 and auxiliary dispenser. 25 microliter of diluted plasma and 250 microliter of plasmin of known activity were added simultaneously to 100 microliter of substrate S-2251. Reaction rate was assayed with the use of two wavelengths, 450/415. Excellent correlation with manual protocol was observed, r=0.985 and Y=0.88X=8.7. Determination of within-day precision of automatic assay for three sample concentrations (Antiplasmin 25%, 100%, 150%) gave C.V. of 1.5%, 1.71% and 4.46% respectively. Thus, authors arrived at the conclusion that 1) α2PI is mainly determined as protesae inhibitor with the use of S-2251, 2) ABA 100 was successfully employed for automatic determination of antiplasmin with the use of simultaneous addition of plasma and plasmin to substrate solution.

Determination of α1-Antitrypsin with A Chromogenic Peptide Substrate Methodology and Clinical Application

1979 ◽  
Author(s):  
I. Witt ◽  
H. Lill ◽  
D. Karitzky ◽  
H. Flad
Keyword(s):  
Reliable Method ◽  
Minute Volume ◽  
Chromogenic Substrate ◽  
Trypsin Activity ◽  
Peptide Substrate ◽  
Hereditary Defect ◽  
The Difference ◽  
Specific Reactions ◽  
Guide Values

α-Antitrypsin (α-AT) is a relatively unspecific inhibitor of proteinases. The co-dominant hereditary defect of the protein can be associated with obstructive pulmonary emphysema in adulthood or with juvenile hepatocirrhosis.A rapid and reliable method for the determination of α1-AT activity with the chromogenic substrate Z-Val-Gly-Arg-pNA has been developed: a standardized trypsin solution of known activity is allowed to react with the plasma or serum sample and the residual trypsin activity determined after incubation. The difference between the initial and residual trypsin activities corresponds to the α1-AT activity. The assay is carried out in tris/HCl-buffer at pH 8.0 (ionic strength=0.15) and 25°C; measurements are taken at 405 nm. Both kinetic and 2-point measurement is possible. The coefficient of variation for day-to-day precision is <5%.The following guide values were determined on 15 healthy probands: 141 ± 15 inhibitor U/ml for plasma, and 130 ± 25 inhibitor U/ml for serum.The minute volume of plasma or serum used precludes non-specific reactions of the substrate with other proteases. Homozygous carriers of this protein defect can be accurately identified by means of this test.

scholarly journals A simple and reliable method for determination of optimum pH in coupled enzyme assays

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 200-203
Author(s):  
Lee Bowman ◽  
Rachael Motamed ◽  
Paul Lee ◽  
Kadijah Aleem ◽  
Astha S Berawala ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Enzyme Assay ◽  
Reliable Method ◽  
Enzyme Assays ◽  
Ph Optimum ◽  
Optimum Ph

Determination of the optimum pH in a coupled enzyme assay poses significant challenges because altering the pH of the reaction mixture can affect the performance of both enzymes. Here, we demonstrate a simple and reliable method to determine the pH optimum for pyruvate kinase using the pyruvate kinase/lactate dehydrogenase coupled enzyme assay. This simple and reliable method can be broadly adapted to determine the pH optimum for various enzymes that are assayed using a coupled enzyme assay.

Determination of α1-Antitrypsin with a Chromogenic Peptide Substrate Methodology and Clinical Application

1979 ◽  
Author(s):  
I. Witt ◽  
H. Lill ◽  
D. Karitzky ◽  
H. Flad
Keyword(s):  
Reliable Method ◽  
Minute Volume ◽  
Chromogenic Substrate ◽  
Trypsin Activity ◽  
Peptide Substrate ◽  
Hereditary Defect ◽  
The Difference ◽  
Specific Reactions ◽  
Guide Values

α1-Antitrypsin (α1-AT) is a relatively unspecific inhibitor of proteinases . The co-dominant hereditary defect of the protein can be associated with obstructive pulmonary emphysema in adulthood or with juvenile hepatocirrhosis. A rapid and reliable method for the determination of α1-AT activity with the chromogenic substrate Z-Val-Gly-Arg-pNA has been developed: a standardized trypsin solution of known activity is allowed to react with the plasma or serum sample and the residual trypsin activity determined after incubation. The difference between the initial and residual trypsin activities corresponds to the α1-AT activity. The assay is carried out in tris/HCl-buffer at pH 8.0 (ionic strength = 0.15) and 25°C; measurements are taken at 405 nm, Both kinetic and 2 point measurement is possible. The coefficient of variation for day-to-day precision is<5%.The following guide values were determined on 15 healthy probands: 141 ± 15 inhibitor U/ml for plasma, and 130 ± 25 inhibitor U/ml for serum. The minute volume of plasma or serum used precludes non-specific reactions of the substrate with other proteases. Homozygous carriers of this protein defect can be accurately identified by means of this test.

Studies on Nature of So-Called Antiplasmin Measured by Chromogenic Substrate S-2251 and its Automatic Determination with ABA 100

1979 ◽  
Author(s):  
M. Kato ◽  
M. Fujimaki ◽  
K. Fukutake ◽  
A. Maki ◽  
M. Hibino
Keyword(s):  
Protease Inhibitors ◽  
Reaction Rate ◽  
Chromogenic Substrate ◽  
Excellent Correlation ◽  
Automatic Determination ◽  
Substrate Solution ◽  
Simultaneous Addition ◽  
Single Radial Immunodiffusion ◽  
At Iii

Using chromogenic substrate S-2251, authors studied on the nature of so-called antiplasmin and on the application of Coatest to automatic determination. As to several protease inhibitors, correlation performed vs single radial immunodiffusion yielded a regression of Y=19.5X-18.6 and r=0.91 for α2 PI;r=-0.15, 0,13, 0.46 0.28 for α2M, α1AT, AT-III and CIINA respectively. Purified protease inhibitors except α2PI did not exhibit immediate inhibition to human plasmin. The automation of chromogenic substrate method was studied with ABA Bichromatic Analyser Model 100 and auxiliary dispenser. 25 microliter of diluted plasma and 250 microliter of plasmin of known activity were added simultaneously to 100 microliter of substrate S-2251. Reaction rate was assayed with the use of two wavelengths, 450/415. Excellent correlation with manual protocol was observed, r=0.985 and Y=0,88X=8.7. Determination of within-day precision of automatic assay for three sample concentrations (Antiplasmin 252, 100%, 150%) gave C.V. of 1.5%, 1.71% and 4.46% respectively. Thus, authors arrived at the conclusion that 1) OfiPI is mainly determined as protesae inhibitor with the use of S-2251, 2) ABA 100 was successfully employed for automatic determination of antiplasmin witli the use of simultaneous addition of plasma and plasmin to substrate solution.

scholarly journals IMMOBILIZATION OF PAPAIN ON CHITOSAN

2010 ◽  
Vol 8 (3) ◽  
pp. 372-376
Author(s):  
Sari Edi Cahyaningrum ◽  
Narsito Narsito ◽  
Sri Juari Santoso ◽  
Rudiana Agustini
Keyword(s):  
Thermal Stability ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Thermal Stability Of

In this study, papain was immobilized on chitosan with Mg(II) cosslinked agent. Studies on free and immobilized papain systems for determination of optimum pH, optimum temperatur, thermal stability and reusability were carried out. The results showed that free papain had optimum pH 6.5 and optimum temperature 55 °C while the immobile papain hadoptimum pH 8 and optimum temperature 80 °C. The thermal stability of the immobilized papain, relative to that of the free papain, was markedly increased. The immobilized papain can be reused for at least six times.   Keywords: papain, immobilization, chitosan

scholarly journals STUDY ON ADSORPTION OF Cd(II) BY CHITOSAN-ALUMINA

2010 ◽  
Vol 6 (3) ◽  
pp. 238-244 ◽  
Author(s):  
Darjito Darjito ◽  
Danar Purwonugroho ◽  
Siti Nasirotun Nisa
Keyword(s):  
Adsorption Capacity ◽  
Aqueous Phase ◽  
Functional Group ◽  
Adsorption Process ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Before And After ◽  
Infrared Spectrophotometer ◽  
Agitation Time

One techniques to reduce the concentration of heavy metal Cd(II) in aqueous solution is adsorption by chitosan. To modify the surface textures and expose the active binding sites, composite biosorbent has been prepared by coating chitosan onto alumina. The aims of this research were to identify the functional group of chitosan-alumina, to characterize adsorption of Cd(II) by using chitosan-alumina adsorbent including optimum pH, optimum agitation time, and to determine the adsorption capacity of the adsorbent. The functional group of chitosan-alumina was identified by infrared spectrophotometer. Determination of the optimum condition was carried out at 40 ppm Cd(II), 125 rpm and 0,1 g adsorbent. Calculation of adsorpted Cd(II) based on its concentration in aqueous phase before and after adsorption process. The concentrations of Cd(II) in aqueous phase after adsorption process  were determined by using Atomic Absorption Spectroscopy (AAS). Identified functional groups of chitosan-alumina were -OH (3466.39 cm-1), -NH amine (1625.15 cm-1), C=O (1703.30 cm-1), and Al-O (1302.07 cm-1). The optimum pH was reached at pH 7, optimum agitation time at 15 minutes, and adsorption capacity of chitosan-alumina was 15.35 ± 0.05 mg/g.   Keywords: adsorption, chitosan-alumina, characterization of adsorption

Levels of platelet factor 3 in citrate plasma and in whole blood as determined by a chromogenic peptide substrate assay

1979 ◽  
Author(s):  
H Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PGE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PGE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PGE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PGE 1/theophylline anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and β-tg are released in parallel.

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