Article

Affinity purification of urinary trypsin inhibitor from human urine.

School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu, P. R. China.
Journal of Separation Science (Impact Factor: 2.59). 11/2011; 35(1):1-6. DOI: 10.1002/jssc.201100781
Source: PubMed

ABSTRACT Affinity protocols for the purification of urinary trypsin inhibitor (UTI) were developed. To imitate the substrate/inhibitor-binding domain (S1 domain) of trypsin and chymotrypsin, the key amino acid residues were composed to sorbents. The sorbents were then subjected to adsorption analysis with UTI. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. The purified enzyme was subjected to SDS-PAGE, trypsin inhibitor activity and peptide map fingerprinting analysis. As calculated, the theoretical maximum adsorption (Q(max)) of two affinity sorbents entitled as S-D-G and S-S-G were 31.7 and 30.1 mg/g, respectively; the desorption constants K(d) of the two sorbents were 8.9 and 18.6 μg/mL, respectively. After the separation of UTI with S-D-G and S-S-G, reducing SDS-PAGE analysis revealed that the protein was a single polypeptide with the mass of ~66 kDa, and the purified proteins were ~95 and 97% pure, respectively; the band on gel was further confirmed with peptide map fingerprinting analysis. Protein and bioactivity recoveries were 1.3 and 75.9% with S-D-G, 1.0 and 70.2% with S-S-G, respectively.

0 Bookmarks
 · 
92 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: A new solid phase microextraction (SPME) fiber based on high-temperature three dimensionally honeycomb layered double hydroxide (TDH-LDH) material is presented. The fiber coating can be prepared easily, it is mechanically stable and exhibits relatively high thermal stability. This study shows that three dimensionally honeycomb layered double hydroxide generated porous morphology. The TDH-LDH material was tested for the extraction of some phenolic and polycyclic aromatic hydrocarbon compounds from aqueous sample solutions in combination with gas chromatography-mass spectrometry (GC-MS). The TDH-LDH fiber contains polar groups and its efficiency for non-polar polycyclic aromatic hydrocarbon compounds was lower than phenolic compounds. On the other hand, a high tendency towards the adsorption of polar phenolic compounds was observed for the proposed fiber. The effects of the extraction and desorption parameters including extraction temperature, extraction time, ionic strength, stirring rate, pH and desorption temperature and time have been studied. In optimum conditions, the repeatability for one fiber (n=5), expressed as relative standard deviation (R.S.D. %), was between 2.8% and 7.1% for the phenolic compounds. The detection limits for the studied phenolic compounds were between 0.02 and 5.8ngmL(-1). The developed method offers the advantage of being simple to use, with shorter analysis time, lower cost of equipment, thermal stability of fiber and high relative recovery in comparison to conventional methods of analysis.
    Journal of Chromatography A 04/2014; · 4.61 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A highly porous fiber coated with polythiophene/hexagonally ordered silica nanocomposite was prepared for solid-phase microextraction. The prepared nanomaterial was immobilized onto a stainless-steel wire for the fabrication of the solid-phase microextraction fiber. Polythiophene/hexagonally ordered silica nanocomposite fibers were used for the extraction of some polycyclic aromatic hydrocarbons from water samples. The extracted analytes were transferred to the injection port of a gas chromatograph using a laboratory-designed solid-phase microextraction device. The results obtained prove the ability of the polythiophene/hexagonally ordered silica material as a new fiber for the sampling of organic compounds from water samples. This behavior is due most probably to the increased surface area of the polythiophene/hexagonally ordered silica nanocomposite. A one at-the-time optimization strategy was applied for optimizing the important extraction parameters such as extraction temperature, extraction time, ionic strength, stirring rate, and desorption temperature and time. Under the optimum conditions, the limit of detection of the proposed method is 0.1-3 pg mL(-1) for analysis of polycyclic aromatic hydrocarbons from aqueous samples, and the calibration graphs were linear in a concentration range of 0.001-20 ng mL(-1) (R(2) >0.990) for most of the polycyclic aromatic hydrocarbons. The single fiber repeatability and fiber-to-fiber reproducibility were less than 8.6 and 19.1% (n = 5), respectively.
    Journal of Separation Science 11/2013; · 2.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Keggin-type heteropoly compounds supported on montmorillonite clays was prepared and used as a highly porous fiber coating material for solid-phase microextraction (SPME). The prepared nanomaterial was immobilized onto a stainless steel wire for fabrication of the SPME fiber. The fiber was evaluated for the extraction of some polycyclic aromatic hydrocarbons (PAHs) from aqueous sample solutions in combination with gas chromatography-mass spectrometry (GC-MS). A one at-the-time optimization strategy was applied for optimizing the important extraction parameters such as extraction temperature, extraction time, ionic strength, stirring rate, and desorption temperature and time. In optimum conditions, the repeatability for one fiber (n = 3), expressed as relative standard deviation (R.S.D. %), was between 5.1 % and 8.4% for the test compounds. The detection limits for the studied compounds were between 0.02–0.05 pg mL−1. The developed method offers the advantage of being simple to use, with shorter analysis time, lower cost of equipment, thermal stability of fiber and high relative recovery in comparison to conventional methods of analysis.
    Journal of Chromatography A 12/2013; · 4.61 Impact Factor