• Journal of Life Science
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• v.11 no.6
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• pp.561-567
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• 2001

To isolate and purify fibrinolytic active substance from Sarcodon aspratus(N $H_4$)$_2$S $O_4$ precipitation, DE52 anion exchange column chromatography, Sephacryl-S 200gel filtration chromatography and Mono S cation FPLC were carried out and the characterizations of the purified enzyme were investigated. The bound active fraction on DE52 anion exchange column chromatography were eluted with 0.2 M NaCI and the fibrionlytic enzyme was purified after following Sephacryl-S200 gel fitration chromatography and Mono S cation EPLC. The specific activity of purified enzyme was 55.2 U/mg protein and increased 11.3 fold comparing crude extract and the yield was 49.5%. 12% SDS-PAGE electrophoresis and gel filtration chromatography revealed that Sarcodon aspratus fibrionloytic enzyme was highly purified and had 29.300 Da molecular weight. Enzyme activity of the purified fibrinolytic enzyme from Sarcodon aspratus was increased on higher pH and was stable until pH 10.5. On temperature dependent stability, the enzyme activity was decrease sharply but remained 25% relative activity on 8$0^{\circ}C$. This enzyme activity was inhibited by heavy metal ion, C $U^{2+}$ and $Co^{3+}$ with 68% and 38%, respectively. And also, the enzyme activity was inhibited with $Ca^{2+}$ chelator EDTA and serine protease inhibitor PMSF. These results from this study suggested that the fibrinolycit enzyme from Sarcodon aspratus is a serine protease and the enzyme activity was increased by $Ca^{2+}$ or $Mg^{2+}$ ion.n.ion.n.

• BMB Reports
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• v.37 no.3
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• pp.298-303
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• 2004

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• Microbiology and Biotechnology Letters
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• v.9 no.4
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• pp.185-190
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• 1981

1. $\alpha$-amylase from B. circulans F-2 was purified with specific activity 55.0 u/mg. protein (about 23 times of the original specific activity) and the yield of 25.5%, by means of corn starch absorption, salting out with ammonium sulfate (80% saturation), gel filtration on Bio-Gel P-100 and DE-32 column chromatography. 2 The purified enzyme showed two closely migrated protin bands on polyacrylamide disc gel electrophoresis, both of which have amylase activity judging from the activity staining of the gel. On SDS-polyacrylamide disc gel electrophoresis, however, the purified enzyme showed a single band suggesting that those two bands are the charge isomers of an amlyase having the slightly different charge. 3. Plot of log mobility of two bands versus polyacrylamide gel concentration according to Hedrick and Smith gave the parallel lines indicating them to be charge isomers. 4. To confirm the action pattern of two enzyme protein bands, each band was separated and was eluted from the gel and eluates were incubated with soluble starch. Oligosaccharide pattern produced by each eluate was examined by paper chromatography. The eluates of two bands showed the same action pattern. 5. The maltohexaose was the only hydrolysis product of soluble starch in the early stage of hydrolysis.

• Journal of Ceramic Processing Research
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• v.20 no.3
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• pp.222-230
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• 2019

Microwave hydrothermal-assisted sol-gel method was employed to synthesize the Fe doped TiO2 photocatalyst. The morphological analysis suggests anatase phase nanoparticles of ~20 nm with an SBET area of 283.99 ㎡/g. The doping of Fe ions in TiO2 created oxygen vacancies and Ti3+ species as revealed through the XPS analysis. The reduction of the band gap (3.1 to 2.8 eV) is occurred by doping effect. The as-prepared photocatalyst was applied for removal of NOx under solar light irradiation. The doping of Fe in TiO2 facilitates 75 % of NOx oxidation efficiency which is more than two-fold enhancement than the TiO2 photocatalyst. The possible reason of enhancement is associated with high surface area, oxygen vacancy, and reduction of the band gap. Also, the low production of toxic intermediates, NO2 gas, is further confirmed by Combustion Ion Chromatography. The mechanism related NOx oxidation by the doped photocatalyst is explained in this study.

• Restorative Dentistry and Endodontics
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• v.48 no.4
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• pp.39.1-39.23
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• 2023

Objectives: This study aimed to investigate the effectiveness of different topical/systemic agents in reducing the damage caused by bleaching gel to pulp tissue or cells. Materials and Methods: Electronic searches were performed in July 2023. In vivo and in vitro studies evaluating the effects of different topical or systemic agents on pulp inflammation or cytotoxicity after exposure to bleaching agents were included. The risk of bias was assessed. Results: Out of 1,112 articles, 27 were included. Nine animal studies evaluated remineralizing/anti-inflammatories agents in rat molars subjected to bleaching with 35%-38% hydrogen peroxide (HP). Five of these studies demonstrated a significant reduction in inflammation caused by HP when combined with bioglass or MI Paste Plus (GC America), or following KF-desensitizing or Otosporin treatment (n = 3). However, orally administered drugs did not reduce pulp inflammation (n = 4). Cytotoxicity (n = 17) was primarily assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on human dental pulp cells and mouse dental papilla Cell-23 cells. Certain substances, including sodium ascorbate, butein, manganese chloride, and peroxidase, were found to reduce cytotoxicity, particularly when applied prior to bleaching. The risk of bias was high in animal studies and low in laboratory studies. Conclusions: Few in vivo studies have evaluated agents to reduce the damage caused by bleaching gel to pulp tissue. Within the limitations of these studies, it was found that topical agents were effective in reducing pulp inflammation in animals and cytotoxicity. Further analyses with human pulp are required to substantiate these findings.

• Proceedings of the Korea Information Processing Society Conference
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• 2008.05a
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• pp.455-458
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• 2008

단백질체학에서 2-DE는 조직내의 단백질을 규명하는 단백질 분리 기술로서 2-DE에 의하여 생성된 단백질 이미지에서 스팟 매칭을 진행하여 상이한 단백질 젤 내에 존재하는 동일한 단백질 클래스를 찾을 수 있다. 그러나 단백질 2-DE 이미지는 실험 환경의 변화에 민감하여 이미지의 위치적인 변형이나 먼지, 공기방울 등으로 인해 많은 에러 정보를 포함할 수 있다. 이러한 에러는 스팟 매칭에 치명적인 영향을 주어 낮은 정확도를 가지게 된다. 본 논문에서는 단백질 2-DE 이미지 분석을 위한 스팟 매칭에서의 정확도를 향상시키기 위하여 기준점 학습과 기준점 추출의 두 단계로 이루어진 자동화된 기준점 추출 방법을 사용하여 스팟 매칭의 정확도를 향상시킬 수 있는 최적의 기준점을 선정하는 방법을 제안하며 선정된 기준점을 기반으로 다수의 기준 이미지를 선택하여 스팟 매칭을 반복적으로 진행함으로써 확률 기반의 정확한 스팟 매칭 결과를 도출하고자 한다. 특히 데이터 마이닝 기법에서 사용되는 최소지지도 값을 적용함으로써 지지도가 높은 스팟 매칭 결과를 빈발한 스팟 매칭으로 판정한다. 제안한 스팟 매칭 정확도 향상 기법의 정확도를 평가하기 위하여 실제 단백질 2-DE 젤 이미지 데이터를 사용하여 입력 기준점의 개수와 최소 지지도의 증가에 따른 정확도의 변화를 분석하였다.

• BMB Reports
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• v.42 no.7
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• pp.450-455
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• 2009

The "stage albinism line of winter wheat" FA85 was a specific natural mutant strain on leaf color. This physiological mutation was controlled by cytogene. In order to reveal the genetic and biochemical mechanism of albinism, 2-DE was used to investigate the difference of chloroplast protein expression pattern between FA85 and its parent wheat Aibian 1. From the results of 2-DE gels analysis, approximately 683 spots were detected on each gel, and 57 spots were expressed differently at least two-fold. Using MALDI-TOF/TOF MS, 14 of 57 spots were identified, which could be categorized into four classes: carbon metabolism, energy metabolism, defense/stress response and signal transduction. Compared with the parent wheat, the expression of ATPase-$\gamma$ and GP1-$\alpha$ was up-regulated in FA85, and of other proteins was down-regulated. Together, we concluded that the expression of chloroplast proteins had changed obviously in FA85, which might be related to the leaf color mutant.

• 한국정보컨버전스학회:학술대회논문집
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• 2008.06a
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• pp.113-116
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• 2008

In this paper we suggest two novel methods for an implementation of the spot detection phase in the 2-DE gel image analysis program. The one is the adaptive thresholding method for eliminating noises and the other is the asymmetric diffusion model for spot matching. Remained noises after the preprocessing phase cause the over-segmentation problem by the next segmentation phase. To identify and exclude the over-segmented background regions, il we use a fixed thresholding method that is choosing an intensity value for the threshold, the spots that are invisible by one's human eyes but mean very small amount proteins which have important role in the biological samples could be eliminated. Accordingly we suggest the adaptive thresholding method which comes from an idea that is got on statistical analysis for the prominences of the peaks. There are the Gaussian model and the diffusion model for the spot shape model. The diffusion model is the closer to the real spot shapes than the Gaussian model, but spots have very various and irregular shapes and especially asymmetric formation in x-coordinate and y-coordinate. The reason for irregularity of spot shape is that spots could not be diffused perfectly across gel medium because of the characteristics of 2-DE process. Accordingly we suggest the asymmetric diffusion model for modeling spot shapes. In this paper we present a brief explanation ol the two methods and experimental results.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.9
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• pp.1296-1302
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• 2004

This study was conducted to evaluate the possible application of 2 D-SDS-PAGE (2 DE)-based proteome analysis techniques to the assessment of extreme proteolysis in postmortem skeletal muscle. Eight Hanwoo longissimus muscles were incubated immediately after slaughter for 24 h at 5$^{\circ}C$, 15$^{\circ}C$ or 36$^{\circ}C$. Warner Bratzler (WB)-shear force and ultrastructural configuration were determined at 24 h, and rate of proteolysis to 24 h was determined by 1 D-SDS-PAGE (1 DE) and 2 DE. In addition, tentative protein identification was performed from peptide mass fingerprints of MALDI-ToF analysis of major protein groups on 2 DE profiles. The result showed that although ultrastructural configuration was similar between the 5$^{\circ}C$ and 36$^{\circ}C$ treatments, meat at 5$^{\circ}C$ had higher WBshear force (approximately 5 kg greater). A higher rate of protein degradation at 36$^{\circ}C$ was observed based on Troponin-T degradation, 1 DE, and 2 DE analysis. This indicates that proteolysis during the early postmortem period was a significant determinant of shear force at 24 h. Little difference in proteolysis between 5$^{\circ}C$ and 15$^{\circ}C$ treatments was found based on classic 1 DE profile assessment. Meanwhile, considerable differences in the 2 DE profiles between the two treatments were revealed, with substantially higher rate of proteolysis at 15$^{\circ}C$ compared to 5$^{\circ}C$. Nuclease treatment improved 2 DE profile resolution. 400 ${\mu}$g and 600 ${\mu}$g of sample loading appeared to be appropriate for 24 cm pH 3-10 and pH 5-7 IPG strips, respectively. Protein detection and quantification of the 5$^{\circ}C$, 15$^{\circ}C$ and 36$^{\circ}C$ 2 DE profiles revealed 78, 163 and 232 protein spots respectively that were differentially modified in terms of their electrophoretic properties between approximately pI 5.3-7.7 with the molecular weight range of approximately 71-12 kDa. The current results demonstrated that 2 DE was a superior tool to 1 DE for characterising proteolysis in postmortem skeletal muscle.

• Korean Journal of Agricultural Science
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• v.45 no.4
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• pp.635-643
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• 2018

Bovine milk is widely consumed by humans and is a primary ingredient of dairy foods. Proteomic approaches have the potential to elucidate complex milk proteins and have been used to study milk of various species. Here, we performed a proteomic analysis using 2-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) to identify whey proteins in bovine milk obtained soon after parturition (bovine early milk). The major casein proteins were removed, and the whey proteins were analyzed with 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The whey proteins (2 mg) were separated by pI and molecular weight across pH ranges of 3.0 - 10.0 and 4.0 - 7.0. The 2-DE gels held about 300 to 700 detectable protein spots. We randomly picked 12 and nine spots that were consistently expressed in the pH 3.0 - 10.0 and pH 4.0 - 7.0 ranges, respectively. Following MALDI-TOF MS analysis, the 21 randomly selected proteins included proteins known to be present in bovine milk, such as albumin, lactoferrin, serum albumin precursor, T cell receptor, polymeric immunoglobulin receptor, pancreatic trypsin inhibitor, aldehyde oxidase and microglobulin. These proteins have major functions in immune responses, metabolism and protein binding. In summary, we herein identified both known and novel whey proteins present in bovine early milk, and our sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed their expression pattern.