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.

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.

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 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

scholarly journals Rapid Micromethod for the Preparation of Leucocyte-Free Haemolysates for the Determination of Pyruvate Kinase and other Erythrocyte Enzymes

1975 ◽  
Vol 12 (1-6) ◽  
pp. 263-268 ◽  
Author(s):  
A. Westwood
Keyword(s):  
High Activity ◽  
Pyruvate Kinase ◽  
Lysosomal Enzyme ◽  
Capillary Blood ◽  
Enzyme Assays ◽  
Pyruvate Kinase Deficiency ◽  
Erythrocyte Enzyme ◽  
Differential Lysis ◽  
Sensitive Marker

In pyruvate kinase deficiency the high activity of the leucocyte isoenzyme may mask the erythrocyte defect. Separation of leucocytes from erythrocytes by the commonly used sedimentation or filtration procedures requires rather large volumes of blood, is time-consuming, and sometimes leaves an unsatisfactorily large proportion of leucocytes. In the present study at least 95% of leucocytes, measured with the lysosomal enzyme β-N-acetylhexosaminidase as a very sensitive marker for leucocyte lysis, were consistently removed by differential lysis of erythrocytes in hypotonic saline. Haemolysates suitable for erythrocyte enzyme assays can be prepared from very small quantities of capillary blood (10–100 μl) very rapidly, cheaply, and reproducibly by this method.

scholarly journals Determination of Purine Nucleoside Diphosphate by use of Pyruvate Kinase and Lactate Dehydrogenase.

1995 ◽  
pp. 86-88
Author(s):  
Hiroshi SHIMOFURUYA ◽  
Tamaki MIZUTANI ◽  
Masashi NAKAMURA ◽  
Jiro Suzuki
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Purine Nucleoside

scholarly journals Pemanfaatan Biomassa Serbuk Gergaji Sebagai Penyerap Logam Timbal

2017 ◽  
Vol 5 (4) ◽  
pp. 166
Author(s):  
Dey Intan ◽  
Irwan Said ◽  
Paulus Hengky Abram
Keyword(s):  
Heavy Metal ◽  
Adsorption Capacity ◽  
Adsorption Process ◽  
Ph Optimum ◽  
Optimum Weight ◽  
Optimum Ph ◽  
High Level

Lead (Pb) is one kind of heavy metal that has high level of toxicity. One way to reduce the level of Pb is by adsorption using cellulose and lignin of sawdust. The aim of this study is to determine the optimum pH, the optimum weight and to determine the adsorption capacity of sawdust when it absorbs Pb in solution of Pb(NO3)2. The adsorption process is carried out by using the various pH of 3, 4, 5, 6, 7, and 8 with a weight of 100 mg, and then the various weight of 100, 200, 300, 400, and 500 mg with the pH optimum. The analysis of Pb content in the solution was conducted by Spectro-direct. The analysis result shows the determination of pH occured at pH 7, Pb absorbed is 14.89 mg/g, and the percentage of Pb absorbed was 96.97%. For the determination of 400 mg of the adsorbent weight of Pb absorbed was 3,83 mg/g, the adsorption percentage of Pb was 99.98%, and the optimum adsorption for optimum weight was 0.15 mg Pb/mg sawdust.

Venous Serum, Capillary Serum, and Capillary Plasma Compared for Use in Determination of Lactate Dehydrogenase and Aspartate Aminotransferase Activities

1975 ◽  
Vol 21 (7) ◽  
pp. 896-897 ◽  
Author(s):  
Russell E Haymond ◽  
Joseph A Knight
Keyword(s):  
Lactate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Enzyme Assay ◽  
Capillary Tube ◽  
Capillary Blood ◽  
Tissue Fluid ◽  
Tube Size

Abstract Lactate dehydrogenase and aspartate aminotransferase activities in capillary serum or plasma were significantly greater than in simultaneously assayed venous serum, the greatest differences being between capillary and venous serum. Although some difference is attributable to tissue fluid contributions, platelets seem to account for most of it, with possible small contributions from leukocytes. Capillary tube size and type appear to be important factors. We recommend that when capillary blood is to be used for enzyme assay, it should be processed as plasma.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

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