• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.87-91
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• 2005

Although 2D-PAGE has been widely used as the primary method for protein separation, difficulties in displaying proteins with an extreme values of isoelectric paint (pI), molecular size and hydrophobicity limit the technique. In addition, time consuming steps involving protein transfer and extraction from the gel-pieces can result in sample loss. Here, we describe a novel protein separation technique with capillary size-exclusion chromatography (CSEC) for rapid protein identification from human plasma. The method includes protein fractionation along with molecular size followed by in-solution tryptic digestion and peptide analysis through reversed phase liquid chromatography (RPLC) coupled to nanoflow electrospray-tandem mass spectrometry (ESI-MS/MS). Tryptic peptides are applied an a $100\;{\mu}m\;i.d.{\times}10mm$ length pre-column and then separated on a $75\;{\mu}m{\times}200mm$ analytical column at -100 nL/min flaw rate. Proteins were identified over the wide ranges of pI (3.7-12.3) when this technique was applied to the analysis of $1-2\;{\mu}L$ of human plasma. This gel-free system provides fast fractionation and may be considered a complementary technique to SDS-PAGE in proteomics.

• KSBB Journal
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• v.15 no.5
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• pp.464-468
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• 2000

The methanol extract of Arachis hypogaea shell showed antioxidative and antimicrobial activity. The methanol extract was successively purified by solvent fractionation, silica gel adsorption column chromatography, Sephadex LH-20 column chromatography and octadecylsilane column chromatography. The purified active substances were isolated by high performance liquid chromatography, and were identified as 3-methoxy-4-hydroxybenzoic acid and 4-hydroxybenzoic acid by LC-MS and GC-MS. The amount of 3-methoxy-4-hydroxybenzoic acid and 4-hydroxybenzoic acid were 3.8mg and 9.8 mg per kg of shell, respectively.

• Applied Biological Chemistry
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• v.39 no.4
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• pp.265-267
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• 1996

The methanol extracts of AraIia elata showed antimicrobial activity against bacteria. The antimicrobial active principle was successively purified with solvent fractionation, silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, silica gel partition column chromatography and HPLC. The active substance was isolated by HPLC on $C_{18}$ column with an acetic acid-MeOH system. The active substance was identified as trans-4-hydroxycinnamic acid by MS, $^1H-NMR\;and\;^{13}C-NMR$.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.257-257
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• 1994

생쥐의 장내세균으로 부터 황산전이 반응을 촉매하는 효소인 sulfotransferase를 생산하는 균주를 분리하였으며 동정결과 Haemophilus속 균주로 확인되어 Haemophilus K-12라 명명하였다. 균의 성장과 효소활성과의 관계를 보면 균은 10시간에서 완전히 성장하였으며 효소활성도 이와 비례하였다. Haemophilus K-12의 배지조성에 따른 sulfotransferase의 생산성을 Brain Heart Infusion 배지에서의 생산성과 비교해보면 탄소원으로는 sucrose가 0.2%농도에서 584%로 가장 좋았으며 질소원으로는 yeast extract가 266%로 가장 좋았다. 공여체로 PNS를 최종농도 1mM로 하여 배지에 첨가하였을때 212%로 가장 높은 효소증가를 보였다. 2가금속이온에 의한 효소증가는 현저하지 않았으며 $Mn^{2+}$이 107%로 가장 높았고$Zn^{2+}$와 EDTA에 의해서는 저해를 받았다. 이상의 결과를 종합하여 균배양을 위한 이상적인 배지조성을 sucrose 0.2%, yeast extract 1%, $Na_2$HPO$_4$ 0.25%, NaCl 0.5%로 결정하였다. 결정된 최적배지에 균을 10L 배양하여 초음파 처리, 원심분리한 것을 70 % ammonium sulfate fractionation, DEAE-cellulose column chromatography를 2회, Hydroxyapatite column chromatography, chromatdfocusing column chromatography, Silica PAE column chromatography, Sephacryl S-300 superfine column chromatography를 행한결과 specific activity가 6.76 umoie/min/mg protein인 효소액을 얻었으며 homogeneous enzyme였다. 이렇게 해서 얻은 효소를 이용하여 수용체 기질 특이성을 측정한 결과 1-naphthol이 phenol을 100%로 하였을 때 233%로 가장 좋았으며 Eubacterium A-44 sulfotransferase의 좋은 기질이었던 p-acetaminophenol, tyramine, 9-phenanthrol은 좋은 기질이 되지 못했다. 이상의 결과로 미루어 보아Haemophilus K-12 sulfotransferase는 지금까지 보고된 bacterial sulfotrasferase와는 다른 효소로 사려되며 효소반응기전의 규명이 이루어지면 산업적 응용이 가능할것으로 사료된다.

• Korean Journal of Food Science and Technology
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• v.27 no.5
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• pp.807-812
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• 1995

The methanol extracts of Aralia elata bark showed antimicrobial activities against bacteria, yeast, fungi. The solvent fractionated acidic fraction that showed the activity was then successively purified with silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, silica gel partition column chromatography, HPLC and TLC. The isolated major active substance was identified as 3,4-dihydroxybenzoic acid by MS, GC-MS, IR, $^{1}H-NMR\;and\;^{13}C-NMR$. The content of 3,4-dihydroxybenzoic acid was 0.869㎎/g in dried bark of Aralia elata.

• Korean Journal of Food Science and Technology
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• v.16 no.3
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• pp.322-328
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• 1984

These experiments were conducted to purify the glucoamylase produced by Rhizopus oryzae. Two forms of glucoamylase (GI and GII) from Phizopus oryzae were purified by $(NH_2)_2SO_4$ fractionation, acetone fractionation and successive column chromatography on DEAE-cellulose and CM-cellulose. The specific activities of GI and GII toward soluble starch were 157.6 U/㎎. protein (37.5 fold of crude extract), and 164.7 U/㎎. protein (39.2 fold of curde extract), respectively, and the yields of them were 4.3% and 3.8%, respectively. The two purified enzymes have shown a single band by polyacrylamide disc gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The protein bands of their electrophoresis gel were revealed to have glucoamylase activity by iodine staining and were proved to be glycoprotein by periodic acid Schiff's staining.

• Journal of Dairy Science and Biotechnology
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• v.18 no.1
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• pp.1-8
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• 2000

This experiment was carried out to study fractionation and physicochemical characteristics of caprine casein. Acid caseins obtained from caprine colostral and normal milk were analyzed by chymosin treatment and fractionated by DEAE-cellulose column chromatography with linear gradient and electrophoresis. Protein, fat and lactose of caprine normal milk were 2.70${\pm}$0.27%, 3.82${\pm}$0.51%, and 4.10${\pm}$0.29%, respectively. More non-protein nitrogen(NPN) was released by chymosin treatment from caprine colostral casein than normal casein. The electrophoretic pattern of caprine casein was not similar to that of bovine casein, Caprine normal casein was fractionated by DEAE-cellulose column chromatography with a 0.08${\sim}$0.18 M NaCl linear gradient into 5 pes with the proportion of 5.27%, 26.07%, 25.97%, 30.40% and 12.29%, respectively. In order to identify the pure fraction, the chymosin-treated caprine normal casein was fractionated by DEAE-cellulosecolumn chromatography with a 0.08${\sim}$0.18 M NaCl linear gradient into 6 peaks with the proportion of 17.06%, 9.10%, 17.85%, 20.11%, 27.03% and 8.85%, respectively.

• Microbiology and Biotechnology Letters
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• v.8 no.2
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• pp.125-133
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• 1980

A microorganism which produced D-glucose isomerase was identified to be similar to Streptomyces antibioticus on the morphological, cultural and physiological characteristics except the spore chain and the utilization of sucrose. D-xylose grown cells of Streptomyces sp. strain K-17 were disrupted by grinding with sea sand. D-glucose isomerase was partially purified with the fractionation by ammonium sulfate, Mn-treatment, DEAE-cellulose column chromatography, DEAE-sephadex (A-50) column chromatography and gel filtration of sephadex G-200. The enzyme was purified about 380 fold with 25 ％ recovery.

• KSBB Journal
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• v.15 no.2
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• pp.150-155
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• 2000

Antioxidative activity of c비ture of Streptomyces sp. BH-405 was investigated. After removal of pellets of Streptomyces sp. B BH-405, antioxidative substances were is미ated and suc$\infty$sively purified from culture of Streptomyces sp. BH-405 by by thin | layer chromatography $\pi$LC) or silica gel column chromatography. The fraction 3 obtained from ethylether fractionation of the C culture appeared highest level of anti oxidative activity as determined by thiocyanate method. Band 2 obtained by further P purification of this fraction showed higher anti oxidation level than that of same concentration of dl- $\alpha$ -tocopherol, butylated h hydroxy anisole (BHA). The band 2 showed higher rate of 1, 1.diphenyl 2-picrylhydrazyl (DPPH) decolorization than dl-$\alpha$-tocopherol. In the rat liver microsomes, band 2 rapidly inhibited lipid peroxidation which was initiated enzymatically by r reduced nicotinamide adenine dinucleotide phosphate (NADPH) or non-enzymatically by Fenton’s reagent. Band 2 inhibited on | lipid peroxidation of mitochondria or the linoleic acid hydro peroxide induced peroxidation system. It is concluded that band 2 obtained by fractionation of Streptomyces sp. BH-405 cultivation contained antioxidants with the capacity to inhibit oxidative m modification.

• Preventive Nutrition and Food Science
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• v.3 no.4
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• pp.334-338
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• 1998

Kale(Brassica oleracea var. acephala) is one of Cruciferous vegetables that is closely related to the wild ancestral form of cabbabe. The ethanol extract of kale which contains the active compoundsss under Salmonella assay system was fractionated with chloroform to collect the nonpolar solvent soluble compounds, and then further fractionation was carried out by silica gel column chromatography. Among kale extracts separated by silical gel column chromatography, the fractions of 4, 5 and 6 exhibited strong antimutagenic activities. The major active compounds from the fraction were identified as chlorophyll derivatives by the analysis with HPLC-fritp-MS. The molecular weights of each chlorophyll derivatives in the sample were acquired from the peaks of positive ion atomosphere pressure chemical ionization (APCI) mas spectrometry.