Kinetic studies of partially purified bromelain from Bogor pineapple (Ananas comosus [L.] Merr) core with ion exchange chromatography and in vitro evaluation of its antiplatelet activity

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

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THE UPTAKE OF TESTOSTERONE AND ZINC IN VITRO BY THE HUMAN BENIGN HYPERTROPHIC PROSTATE

Journal of Endocrinology ◽

10.1677/joe.0.0580405 ◽

1973 ◽

Vol 58 (3) ◽

pp. 405-419 ◽

Cited By ~ 27

Author(s):

M. JOAN REED ◽

S. R. STITCH

Keyword(s):

Ion Exchange ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Zinc Binding ◽

Supernatant Fraction ◽

Low Molecular Weight ◽

Molecular Weight Peak ◽

Steroid Binding

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

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Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

10.7287/peerj.preprints.26803v1 ◽

2018 ◽

Author(s):

Bingyu Ye ◽

Wenlong Shen ◽

Minglei Shi ◽

Yan Zhang ◽

Cunshuan Xu ◽

...

Keyword(s):

Ion Exchange ◽

Clinical Investigation ◽

Affinity Purification ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Size Exclusion ◽

Radioprotective Activity ◽

Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

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False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

Clinical Chemistry ◽

10.1093/clinchem/26.2.0345 ◽

1980 ◽

Vol 26 (2) ◽

pp. 345-347

Author(s):

G P James ◽

M H DJang ◽

H H Hamilton

Keyword(s):

Ascorbic Acid ◽

Ion Exchange ◽

Exchange Resin ◽

Anion Exchange Resin ◽

False Negative ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Negative Results ◽

False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

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Inhibitors of Fibrinolysis in Amniotic Fluid

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650075 ◽

1980 ◽

Vol 44 (01) ◽

pp. 032-034 ◽

Cited By ~ 3

Author(s):

J E Walker ◽

D M Campbell ◽

D Ogston

Keyword(s):

Ion Exchange ◽

Amniotic Fluid ◽

Inhibitory Activity ◽

Major Part ◽

Competitive Inhibition ◽

Kinetic Studies ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Porcine Heart ◽

Esterolytic Activity

SummaryAmniotic fluid inhibited the fibrinolytic, amidolytic and esterolytic activity of urokinase. Kinetic studies with AGLMe demonstrated non-competitive inhibition. A major part of the inhibitory activity could be separated from α1-antitrypsin by ion-exchange chromatography on DEAE-Sephadex A-50. Plasminogen activator prepared from porcine heart was not inhibited by amniotic fluid. Amniotic fluid also inhibited the caseinolytic and amidolytic activities of plasmin.

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In Vitro Anticoagulant Activity of Esters of Heparin.

10.1055/s-0039-1687054 ◽

1979 ◽

Author(s):

J. Mardiguian ◽

M. Trillou ◽

J. Marin

Keyword(s):

Molecular Weight ◽

Ion Exchange ◽

Chemical Modification ◽

Anticoagulant Activity ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Hydroxyl Groups ◽

Oh Groups

Chemical modification of Heparin by esterification of its hydroxyl groups has been carried out in order to study its influence on the in vitro anticoagulant activity. Beef and pig mucosal Heparins have been fractionated by gel filtration and by ion-exchange chromatography to yield fractions which have been esterified.Two types of esters have been prepared : 4-chlorophenoxy-acetic and 4-chlorophenoxy-isobutyric.The anticoagulant activity of the these esters has been evaluated in vitro (using U.S.P. method, anti-Xa and anti-thrombin assays) and correlated with their chlorine content and U.V. absorption.This study shows that the in vitro anticoagulant activity of heparin is decreased by esterification of its OH groups, to an extent depending upon the type of ester, the molecular weight and the degree of substitution.The results of these experiments will be reported and discussed in detail.

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Purification, characterization, and bioassay of putative protease inhibitors from Hevea brasiliensis latex

E-Journal Menara Perkebunan ◽

10.22302/iribb.jur.mp.v84i2.220 ◽

2017 ◽

Vol 84 (2) ◽

Author(s):

Riza Arief PUTRANTO ◽

. SISWANTO ◽

Agustin Sri MULYATNI ◽

Asmini BUDIANI ◽

Radite TISTAMA

Keyword(s):

Ion Exchange ◽

Protease Inhibitor ◽

Protease Inhibitors ◽

Hevea Brasiliensis ◽

Rubber Tree ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Sds Page ◽

Putative Protease

Lateks yang menyerupai cairan susu putih diperoleh dari penyadapan kulit batang tanaman karet (Hevea brasiliensis). Lateks merupakan sitoplasma dari jaringan pembuluh bernama latisifer yang didalamnya terkandung berbagai macam komponen, termasuk protein-protein penting. Berbagai jenis enzim yang memiliki fungsi terkait pertahanan tanaman dari serangan patogen dan pelukaan telah berhasil dideteksi di dalam lateks, di antaranya protease inhibitor (PI). Protease inhibitor memiliki aktivitas senyawa antifungi sehingga berpotensi untuk dimanfaatkan sebagai biofungisida. Pada penelitian ini, protease inhibitor putatif yang berasal dari serum B (lutoid) lateks tanaman karet telah berhasil diisolasi menggunakan teknik Ion Exchange Chroma-tography. Dari total 70 fraksi protein yang diekstrak dari kolom, hanya 26 fraksi yang menunjukkan kadar protein yang terukur. Kandungan protease inhibitor putatif yang di-peroleh berkisar antara 0,0067 hingga 0,022 mL/g serum B dari hasil 3 fraksi terpilih. Aktivitas penghambatan terhadap empat enzim protease (subtilisin A, tripsin, α-kimotripsin, dan papain) menunjukkan karakteristik protease inhibitor putatif tersebut sebagai serine dan/atau cysteine inhibitor protease dengan persentase hambatan di atas 15% terhadap protease target. Hasil SDS-PAGE memperlihatkan pemisahan protein dominan yang diperkirakan merupakan protease inhibitor putatif dengan berat molekul sebesar 21,5 kDa. Uji bioassay aktivitas antifungi secara in vitro dari protease inhibitor memperlihatkan penghambatan pertumbuhan miselium dari fungi Ganoderma boninense, Sclerotium sp., dan Rigidosporus lignosus. [Kata kunci : protease inhibitor, Hevea brasiliensis, lateks, serum B, ion exchange chromatography]AbstractLatex, a milky white liquid, is the main product from rubber tree (Hevea brasiliensis). Latex is the cytoplasm of complex cellular networks named laticifers in which it contains many different components, including important proteins. Various types of enzymes carrying functions associated with plant defense against pathogen and wounding have been detected in latex in which one of these enzymes is protease inhibitor (PI). Plant protease inhibitor has tremendous potential as an antifungal agent which can be developed as biofungicide. In this work, protease inhibitors from B-serum (lutoid) of rubber tree latex were isolated and purified using Ion Exchange Chromatography (IEC) technique. Of the total 70 fractions of proteins extracted from the columns, only 26 fractions showed measurable levels of protein. The concentration of obtained putative protease inhibitors (three fractions of IEC) ranged from 0.007 to 0.022 mL/g B-serum. Inhibitory activity against four protease enzymes (subtilisin A, trypsin, α-chymotrypsin, and papain) showed the characteristics of Hevea putative protease inhibitors from B-serum as serine and/or cysteine protease inhibitors with more than 15% inhibitory activity of target protease. Based on SDS-PAGE visualization, the molecular weight of dominant protein considered as Hevea putative protease inhibitors was 21.5 kDa. In vitro bioassay test of antifungal activity for Hevea putative protease inhibitors showed reduced mycelium growth of Ganoderma boninense, Sclerotium sp., and Rigidosporus lignosus.[Keywords: protease inhibitor, Hevea brasiliensis, latex, B-serum, ion exchange chromatography]

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