In three-phase partitioning (TPP) ammonium sulphate and 2-methyl-2-propanol (tert-butanol) are used for separation of proteins by collecting and concentrating them into the middle layer or third phase of the system. During the last two decades, TPP has been primarily applied to enzyme purification at laboratory scale, however, its analytical type application for investigating meat has also been reported. Here, an experimental set of TPP feasible to analytical scale experiments is described to study partitioning behaviour of carp and corn proteins. In partitioning experiments, corn proteins extractable with borate buffer, and sarcoplasmic proteins of carp were investigated. In the pH-range of 4.8-7.9, composition of partitioned corn proteins showed independence of pH as it was indicated by nonequilibrium pH gradient electrophoresis (NEpHGE) patterns. In case of carp, the alterations of protein composition in the pH-range of 3.7-8.0 unambiguously indicated the pH-dependent partitioning of carp proteins. The analytical scale method, based on three-phase partitioning is a new approach to investigate food proteins.
The Wizard Clean up System and a three-phase partitioning (TPP) method were used to purify genomic pork-DNA of various food samples for amplification. Quality of DNA purified by Wizard resin and partitioning was controlled by spectrophotometer and electrophoresis, respectively. A 108 bp fragment from the porcine growth hormone gene was applied, according to M EYER and co-workers (1994). Of all the samples prepared, amplicons were obtained by the pork- DNA specific PCR. Partitioning was found to be an efficient DNA purification step in preparation of PCR-grade DNA.
A novel step in bean (Phaseolus vulgaris) α-amylase inhibitor (AAI) purification, based on the application of an inorganic adsorbent, zinc hydroxide, was developed. The new method was substantially faster than existing protocols. Up to 98% of bean seed proteins were bound to the white precipitate in the range of 1–4% (w/v) zinc hydroxide, while the amount of bound bean AAI was far less in the range of 1–2%. The AAI-enriched fraction, unbound by zinc hydroxide, was further purified by DEAE-(diethylaminoethyl)chromatography and gel filtration. It was found that zinc hydroxide binds the majority of soluble proteins of bean, while it leaves α-amylase inhibitor in solution. The binding of proteins to zinc-hydroxide occurs in a short time and the change caused in the buffer composition is insignificant, thus it may open new approaches in purification of other proteins, too.
The main goal of our work was to develop a rapid, simple, and economical DNA extraction method for food (especially for meat products) analysis. This extraction and purification procedure was based on the three-phase partitioning (TPP) method. The developed new DNA-TPP method and Wizard DNA Clean-Up System (Promega, USA) have been compared concerning extraction efficiency, purity and DNA suitability for amplification. The quality and quantity of the purified DNA solutions were controlled by spectrophotometer and the amplification efficiency by simple qualitative PCR. All of prepared DNA solutions were pure enough for the PCR and contained appropriate quantity of DNA. Thus, 118 bp length amplicons could have been obtained by the specific lectin-gene PCR in all cases. This method proved to be an alternative one to isolate DNA from meat samples simply and economically.
The aim of this study was to develop an accurate, fast and safe routine diagnostic method based on protein studies for differentiating between T. caries and T. controversa at species level. Since import of wheat contaminated with T. controversa is restricted by several countries, differentiation of T. controversa from the more prevalent T. caries is of economic interest. The newly developed method is based on distilled water washing followed by the rupturing of the teliospore walls in PBS extraction medium, and an SDS electrophoresis (10% resolving gel). The electrophoretic pattern showed consistent species-related differences in a 106 kDa polypeptide that appeared in each extract of T. controversa, but was not present in the protein extracts of T. caries. The newly developed method could be of value for the authorities performing routine monitoring of T. controversa as an up-to-date diagnostic assay in wheat shipments.
Authors:N. Adányi, E. E. Szabó, M. Váradi, and J. Szamos
To explore new possibilities of enzyme immobilization, we investigated bioactive layers prepared by a new procedure based on three-phase partitioning (TPP) of proteins. By this method a third phase or midlayer as a protein layer can be developed at the interface of a protein system containing two phases (organic solvent/aqueous salt solution). Proteins of meat origin partitioned together with bioselective material (e.g. an enzyme) after centrifugation resulted in excellent bioactive layers.In the newly developed sensor, glucose oxidase was immobilized in a layer, which was fixed on the surface of a platinum ring electrode. The biosensor was built in a flow injection analyzer (FIA) system, where the hydrogen peroxide generated during the enzymatic reactions was determined by an amperometric cell. The parameters for biochemical and electrochemical reactions (ion concentration and pH of buffer, flow rate) were optimized. The linear range of analysis by the newly developed sensor was from 0.5 to 10 mmol l–1 glucose. The biosensor could be used for more than 300 analysis.
Authors:M. Novák-Hajós, J. Szamos, M. Gasztonyi, and Gy. Hajós
Seed protein pattern of control and M
mutant soybean (
[L.] Merr.) lines in defatted and non-defatted raw flour was studied after 60% 2-propanol extraction, SDS-PAGE separation, colloidal staining and densitometric evaluation to detect a new variant of the protein KTI and/or BBI, furthermore to find new protein(s) of low molecular weight. Electrophoretic separation of defatted and non-defatted control soybean samples showed the same protein patterns. On the densitograms of mutant lines quantitative and qualitative differences could be observed. Defatted raw soy samples reflected more differences in the number of peaks than non-defatted ones. Beside soy trypsin inhibitors, several more soy proteins of low molecular weight are dissolved. KA mutant line 9 has a unique 2-propanol soluble protein pattern, and a new protein band of Rf=0.37 compared to the control line. Sixty percent 2-propanol soluble soybean seed proteins are suitable for cultivar identification and characterization, furthermore to distinguish soybean lines of the same origin.