CS206378B1 - Method of isolation and purification of the lactatedehydrogenasis - Google Patents
Method of isolation and purification of the lactatedehydrogenasis Download PDFInfo
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- CS206378B1 CS206378B1 CS800179A CS800179A CS206378B1 CS 206378 B1 CS206378 B1 CS 206378B1 CS 800179 A CS800179 A CS 800179A CS 800179 A CS800179 A CS 800179A CS 206378 B1 CS206378 B1 CS 206378B1
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- 238000000034 method Methods 0.000 title claims description 12
- 238000002955 isolation Methods 0.000 title claims description 4
- 238000000746 purification Methods 0.000 title claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 23
- 239000001913 cellulose Substances 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 10
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 238000011067 equilibration Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000012620 biological material Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 claims description 4
- 108010088751 Albumins Proteins 0.000 claims description 4
- 102000009027 Albumins Human genes 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 229960000643 adenine Drugs 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 230000003197 catalytic effect Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims 1
- 229930024421 Adenine Natural products 0.000 claims 1
- 108010020056 Hydrogenase Proteins 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims 1
- 229960003966 nicotinamide Drugs 0.000 claims 1
- 235000005152 nicotinamide Nutrition 0.000 claims 1
- 239000011570 nicotinamide Substances 0.000 claims 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 8
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 8
- 239000000975 dye Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 150000003918 triazines Chemical class 0.000 description 4
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- YKCWQPZFAFZLBI-UHFFFAOYSA-N cibacron blue Chemical compound C1=2C(=O)C3=CC=CC=C3C(=O)C=2C(N)=C(S(O)(=O)=O)C=C1NC(C=C1S(O)(=O)=O)=CC=C1NC(N=1)=NC(Cl)=NC=1NC1=CC=CC=C1S(O)(=O)=O YKCWQPZFAFZLBI-UHFFFAOYSA-N 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- YZEUHQHUFTYLPH-UHFFFAOYSA-N 2-nitroimidazole Chemical compound [O-][N+](=O)C1=NC=CN1 YZEUHQHUFTYLPH-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- KUIXZSYWBHSYCN-UHFFFAOYSA-L remazol brilliant blue r Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(N)=C2C(=O)C3=CC=CC=C3C(=O)C2=C1NC1=CC=CC(S(=O)(=O)CCOS([O-])(=O)=O)=C1 KUIXZSYWBHSYCN-UHFFFAOYSA-L 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
Description
(54) Sp6sob izolácie a purifikácie laktátdehydrogenázy(54) A method for isolating and purifying lactate dehydrogenase
Vynález sa týká jpdsobu izolácie a purifikácie laktátdehydrogenázy pomocou řarebných Remazolových a triazínovýeh derivátov perlovej celulózy.The invention relates to a process for the isolation and purification of lactate dehydrogenase using colored Remazol and triazine derivatives of pearl cellulose.
Triazínové farbivá, a to hlavně Cibacron Blue F3G-A, kovalentne viazané na vodonerozpustné polysaeharidy, našli v posledněj době široké možnosti uplatnenie ako generálne afinanty v afinitnej chromatografii enzýmov, a to najma zo skupiny dehydrogenáz a kináz. £Ř. S. Beiesner, F. B. Rudolph, J. Chromatogr. 161, 12? /1978/J. NajvSčšie uplatnenie tu našli polysaeharidy na báze sieťovanej· agarózy vo formě komerčných preparátov Blue Sepharose®Triazine dyes, especially Cibacron Blue F3G-A, covalently bound to water-insoluble polysaeharides, have recently found wide application as general affinents in enzyme affinity chromatography, in particular from the dehydrogenase and kinase family. £ r. Beiesner, S. Rudolph, F. Chromatogr. 161, 12? / 1978 / J. Cross-linked agarose-based polysaeharides have been found to be particularly useful here in the form of commercial Blue Sepharose® preparations.
CL-6B (Pharmacia Fine Chemicals, Uppsala; 275,- Sw. Grs. per 25 g), Af f i-Gel ® Blue (Bio·· <* TM TffiT'·'CL-6B (Pharmacia Fine Chemicals, Uppsala; 275, - Sw. Grs. Per 25 g), Af fi-Gel ® Blue (Bio ·· <* TM TffiT '·'
Rad Laboratories, Munchen; 220,- Dlí.per 100 ml) či Matrex Gel Blue A a Matrex Gel Sed A (Amiccn B. V., Oosterhout). Aj iné polysaeharidy, napr. sieťované dextrány, vykazujú dobré vlastnosti pre tento druh použitia. Nie je tomu tak u celulózy, ktorá sa doteraz jevila ako úplné nevhodná (H.-J. Bohme et al., J. Chromatogr. 69, 209 (1972); S. Angal, Ρ» D. G. Dean, Biochem. J. 167, 301 (1977)· Negativnou vlastnosťou tohoto polysacharidu derivatizevaného farbivom Cibacron Blue F3G-A boli velmi nízké resorbcie chromátografováných enzýmov.Rad Laboratories, Munich; 220, - Dlper 100 ml) or Matrex Gel Blue A and Matrex Gel Sed A (Amicin B.V., Oosterhout). Other polysaeharides, e.g. crosslinked dextrans, show good properties for this kind of use. This is not the case with cellulose, which has so far appeared to be completely inappropriate (H.-J. Bohme et al., J. Chromatogr. 69, 209 (1972); S. Angal, DG Dean, Biochem. J. 167 , 301 (1977) · The negative property of this Cibacron Blue F3G-A derivatized polysaccharide was the very low resorption of chromatographed enzymes.
Ako ame už predtým ukázali, triazínové a Reaazolové deriváty perlovej celulózy si zachovávajú znamenité vlastnosti perlovej celulózy. Naviac pri správnéj volbě afinanta je možné překonat všetky nevýhody doteraz brániace použitiu celulózy na tieto účely.As we have previously shown, the triazine and reaazole derivatives of pearl cellulose retain the excellent properties of pearl cellulose. Moreover, with the correct choice of affinity, it is possible to overcome all the disadvantages hitherto preventing the use of cellulose for these purposes.
Podstata vynálezu spočívá v tom, že sa na perlovú celulózu a obsahom 50 až 100 pmólovThe essence of the invention consists in the preparation of pearl cellulose containing 50 to 100 pmoles
206 378206 378
-* ' 2· kovalentne viasaného Reaazolového alebo triazínového farbiva na 1 g sochej perlovej celulózy nechá v prvom stupni pfisobiť zriedený roztok hovBdzieho sérového albuminu, prebytočný albumin sa odstráni prenytím ι 1 I NaCl a po equilibrovahi tlmivým roztokem o pH 6,5 eí 8 aa v druhom stupni na takto upravená perlová celulózu nechá pDaobit homogenizovaný biologický materiál obsahujáci laktátdehydrogenázu, pričom vžeťky sprievodné nežiadáce nízkoa vysokomolekulové látky sa odstránia následnou eláciou a rovnakým equilibračnýa roztokom a potom equilibračnýa roztokom obsahujdeim 0,5 ti 1 K nikotíneeid-adenín-dinukleotid vždy dovtedy, kým nie je reakcia eluátu na bielkoviny negativna, načo v tretom stupni sa adsorbovaný, prečiatený enzým laktátdehydrogenáza vytěsní eláciou s equilibračnýa roztokom obsahujáci® 0,5 až 1,5 mM redukovaný nikotínamid-adenín-dinukleotid dovtedy, kým nie j® skážka eluátu na katalytická aktivitu laktátdehydrogenázy negativna a nakoniec aa perlová celulóza připraví na opakované použiti® premytím s 1 M NaCl do negatívnej reakcie premývacieho roztoku na bielkoviny.The covalently bound Reaazole or triazine dye per 1 g of sculpted pearl cellulose is treated in the first step with a dilute solution of bovine serum albumin, the excess albumin is removed by washing with 1 L NaCl and equilibrating with buffer at pH 6.5 e. in a second step, the treated bead cellulose is then treated with a homogenized biological material containing lactate dehydrogenase, wherein all of the accompanying undesirable low and high molecular weight substances are removed by subsequent elution with the same equilibration solution and then with an equilibration solution containing 0.5 thiucleotide until the reaction of the eluate to the proteins is negative, in the third step the adsorbed, purified lactate dehydrogenase enzyme is displaced by elution with an equilibration solution containing 0.5-1.5 mM reduced nicotinamide adenine dinucleotide until the eluate is blocked for catalytic and lactate dehydrogenase activity is negative and finally pearl cellulose is prepared for reuse® by washing with 1 M NaCl until the protein washing solution has a negative reaction.
Perlová celulóza, ako aj jej Remažolové či triazínové deriváty obaahujáce elM NaCl nevyaytelný albumin nejavia žiadne nespecifické aorpcie voči laktátdehydrogenáze (IBM).Pearl cellulose, as well as its Remazol or triazine derivatives containing an ElM NaCl non-detectable albumin, exhibit no non-specific lactate dehydrogenase (IBM) absorption.
Pre sfinitná chroaatografiu LDH z biologického materiálu .nie je vhodný Cibaeren SLue F3G-A. Heci toto farbivo má ekutočne vysoká afinitu ku LDH, jeho derivát perlovéj celulózy stále prejavuje nízku áčinnosť resorbcie.Cibaeren SLue F3G-A is not suitable for Sphinite LDH chromatography from biological material. Although this dye has a truly high affinity for LDH, its pearl cellulose derivative still exhibits low resorption activity.
Pre popíaaný jednostupnový vysokoúčinný izolačný postup je možné použit hrubé homogenáty z rdznych biologických materiálov počnúc kvasničnýai extraktem! až po Specializované orgány cicavcov, napr. sval, pečen, srdce a pod. Nevhodným je vySSÍ obsah sérových bielkovín.For the described single-step high-performance isolation process, coarse homogenates of various biological materials can be used starting with the yeast extract! up to Specialized mammalian organs, e.g. muscle, liver, heart and the like. Higher serum protein content is inappropriate.
Jefineduchším © lacnejSía je pestup, pri ktorom ea premýva derivatizovená perlová celulóza ao zachytanou LDH equilibračnýa tlmivým roztokom obaahujáeim 1 mM redukovaný aiketínamid-adenín-dinukleotid (NADH). Sluát obsahuje viac než 90 % dávkovanéj LDH s minimálně 15-náaobným stupnom purifikácie. Naproti tomu keS aa eluuje tlmivým roztekom ebaahujáeim z 1 mM NADH po predchádzajáeej eláeii a 1 mM nikotínamid-adenín-dinukleetidem (NAD), eluát / obsahuje aenšie množstvo ensýau, avšak viac než 25-násobne purifikováného.The most cost-effective method is a puff in which the wash washes the derivatized pearl cellulose and contains the captured LDH equilibration buffer containing 1 mM reduced aiketin-adenine dinucleotide (NADH). The acetate contains more than 90% of the dosed LDH with at least a 15-degree purification degree. In contrast, when eluting with a buffer span from 1 mM NADH after previous elution and 1 mM nicotinamide adenine dinucleetide (NAD), the eluate contains an even greater amount of purified but more than 25-fold purified.
. · . i . Nakoniec ták, ako Cibacron Blue P3G-A deriváty sieťovanýeh agarez a dextránov aj Reaazolové a triazínové deriváty perlovej celulózy je možné pre popíaaný postup izoláoie a purifikácie LDH použit viaenásobne.. ·. i. Finally, both Cibacron Blue P3G-A cross-linked agarez and dextran derivatives and the beaded cellulose reaazole and triazine derivatives can be used multiple times for the described procedure for isolating and purifying LDH.
Výhodou navrhovaného postupu je, že umožňuje izolovat a purifikovatThe advantage of the proposed process is that it allows to be isolated and purified
- LDH poměrně jednoduchým technologickým paetupo®,- LDH by relatively simple technological paetupo®,
-LDH na podstatné lačnějších materiálech ako doteraz,-LDH on substantial, more greedy materials than before,
- LDH s vySšču resorbeiou a při vyšších vazbových kapacitách farebnej perlovej celulózy než bole tomu doteraz u známých triazínovýeh derivátev polysaeharidov,- LDH with higher resorption and higher binding capacities of colored pearl cellulose than previously known for the known triazine derivatives of polysaeharides,
- aáčasne viacero enzýmov.- and several enzymes at the same time.
Příklad 1 - .Example 1.
ml Remazol Briliant Blue RN derivátu perlovej celulózy obaahujáceho 65 pmólov farbiva η® 1 g suchej celulózy sa premyl.o s 25 ml 0,1 % roztoku hovádzieho sérového albuminu, 100 ml 10 mil Tris-HSl pufru pH 7,5 e přídavkem 1 mM Chelatonu 3 a 2 mlí 2-merkaptoetanolu (roztok 1), Sálej 100 ml 1 M NaCl a nakoniec 200 ml roztoku 1. Na takto připravený štipec sa nanieslo 25 ml homogenátu z krysých pečení obsahujúceho 310 mg bielkovínml Remazol Brilliant Blue RN pearl cellulose derivative containing 65 pmoles of dye η® 1 g dry cellulose was washed with 25 ml 0.1% bovine serum albumin solution, 100 ml 10 ml Tris-HS1 buffer pH 7.5 e with 1mM Chelatone 3 and 2 ml of 2-mercaptoethanol (solution 1), 100 ml of 1 M NaCl and finally 200 ml of solution 1. 25 ml of rat liver homogenate containing 310 mg of protein were applied to the thus prepared column.
Λ a 473 jednotiek LDH a premylo 130 ml roztoku 1. Naviazaná LDH eajvytesnila 110 ml roztoku 1 s přídavkem 1 mM NADH (roztok 2).And 473 units of LDH and washed with 130 ml of solution 1. Bound LDH e displaced 110 ml of solution 1 with the addition of 1 mM NADH (solution 2).
NADH-frakeia obsahovala 16-násobne prečistenú LDH v množstve 430 jednotiek, čo představuje 98 % na štipec nanesenej LDH.The NADH fractionia contained 16-fold purified LDH at 430 units, representing 98% per column of loaded LDH.
Příklad 2Example 2
Postup podlá příkladu 1 s tým rozdielom, Že sa použil Ostazín červen St5B derivát perlovej celulózy obsahujúci 80 pmólov farbiva na 1 g autíhej celulózy.The procedure of Example 1, except that the Ostazine June St5B bead cellulose derivative containing 80 pmoles of dye per g of cellulose pulp was used.
NADH-frakcia obsahovala 14,5-náaobne prečistenú LDH v množatve 468 jednotiek, čo představuje 99 % na stípec nanesenej LDH.The NADH fraction contained 14.5-naphtha purified LDH in an amount of 468 units, representing 99% per column of loaded LDH.
Příklad'3Příklad'3
Postup podl’a příkladu 1 s tým rozdielom, že sa použil Prócion Biu® MX-B derivát perlovej celulózy obsahujúci 67 pmólov farbiva na 1 g suchéj celulózy.The procedure of Example 1 was followed except that a Prócion Biu® MX-B pearl cellulose derivative containing 67 pmoles of dye per g dry cellulose was used.
liADH-frakcia obsahovala 10,5-násobne prečistenú LDH v množstva 416 jednotiek, čo činilo 88 % na stípec nanesenej LDH.The 11ADH fraction contained 10.5-fold purified LDH at 416 units, which was 88% per column of loaded LDH.
Příklad 4Example 4
Postup podlá příkladu 1 s tým rozdielom, Že po nanesení homogenátu (320 mg bielkovín, 452 jednotiek) sa stípec premýval 250 ml roztoku 1, 100 ml roztoku 1 s prídavkom 1 mM NAD a nakoniec 140 ml roztoku 2.The procedure of Example 1, except that after the homogenate (320 mg protein, 452 units) was applied, the column was washed with 250 ml of solution 1, 100 ml of solution 1 with the addition of 1 mM NAD and finally 140 ml of solution 2.
NADH-frakcia obsahovala 23,5-násobne prečistenú LDH v množstve 266 jednotiek (8 mg bielkovín), čo zodpovědělo‘60 % na stípec nanesenej LDH.The NADH fraction contained 23.5-fold purified LDH in an amount of 266 units (8 mg of protein), corresponding to ‘60% per LDH loaded column.
Příklad 5 ' 'Example 5 ''
Postup podl’a příkladu 1 s tým rozdielom, že sa použilo 15 ml čiastočne přečištěného preparátu LDH z králičieho svalu obsahujúceho 11 mg bielkovín a 440 jednotiek LDH. 'The procedure of Example 1 except 15 ml of a partially purified rabbit muscle LDH preparation containing 11 mg protein and 440 units LDH were used. '
NADH-frakcia obsahovala 100 % na stípec nanesenej LDH.The NADH fraction contained 100% per column of loaded LDH.
Vynález má uplatnenie všade, kde je potřebné izolovať alebo přečistil? laktátdehydrogenázu z rfizneho biologického materiálu od Salších bielkovín, resp. enzýmov, a to v priemyselnej praxi alebo vo výskume.The invention has application wherever it is necessary to isolate or purify? lactate dehydrogenase from a further biological material from other proteins, respectively. enzymes, either in industrial practice or in research.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS800179A CS206378B1 (en) | 1979-11-21 | 1979-11-21 | Method of isolation and purification of the lactatedehydrogenasis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS800179A CS206378B1 (en) | 1979-11-21 | 1979-11-21 | Method of isolation and purification of the lactatedehydrogenasis |
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| Publication Number | Publication Date |
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| CS206378B1 true CS206378B1 (en) | 1981-06-30 |
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| CS800179A CS206378B1 (en) | 1979-11-21 | 1979-11-21 | Method of isolation and purification of the lactatedehydrogenasis |
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| CS (1) | CS206378B1 (en) |
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1979
- 1979-11-21 CS CS800179A patent/CS206378B1/en unknown
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