ABSTRACT
OBJECTIVE：To establish the method for content determination of 17 kinds of amino acids in Sargassum and its adulterants，and to carry out cluster analysis ，so as to provide reference for quality control of Sargassum. METHODS ：Totally of 18 batches of sample （S1-S6 as certified product ，S7-S18 as adulterants ）were collected. After acid hydrolysis ，amino acids contents were detected by using automatic amino acid analyzer. The separation was performed on LCAK 06/Na sulfonic acid cation exchange resin column with mobile phase consisted of buffer-regeneration system （gradient elution ）at the flow rate of 0.45 mL/min （elution pump ）and 0.25 mL/min（derivative pump ）. The detection wavelengths were set at 440 nm（proline）and 570 nm（other amino acids ），and the sample size volume was 50 μL. PASW Statistics 18.0 software was used ，and cluster analysis was conducted by using group connection method of cluster analysis with “square Euclidean distance ”as the measurement standard. RESULTS ：17 kinds of amino acids were well separated without interference from blank sample. The linear relationship between mass concentration and peak area was good （all r were over 0.998），and the upper and lower limits of the linear range were 48.06 μg/L （cystine）and 1.501 μg/L（glycine），respectively；RSDs of precision ，reproducibility and stability tests were lower than 2％. The average recoveries were between 90.60％-101.56％（RSDs were 0.88％-2.15％，n＝6）. 17 kinds of amino acids were detected in Sargassum and its adulterants ，among which the contents of glutamic acid ，aspartic acid ，leucine，alanine，glycine and valine were relatively high . Results of cluster analysis showed that 18 batches of sample were clustered into 4 categories，i.e. S 1-S6 into one category；S7-S9 into one category ；S10-S12，S16-S18 into one category ；S13-S15 into one category ；which was consistent with the identification result of Sargassum and its adulterants . CONCLUSIONS ：The method is simple ， rapid， accurate and reproducible，and can be used for the quantitative analysis and identification of amino acids in Sargassum and adulterants.
ABSTRACT
Objective: To compare the contents of protein and amino acids in different parts of Cervi Cornu Pantotrichum (CCP) with different processing methods, in order to provide a guidance for the processing and comprehensive utilization of CCP. Methods: The techniques of Dumas combustion and cation-exchange chromatography were respectively adopted to determinate the contents of crude protein and 17 amino acids in different parts of CCP with different processing methods, and the difference was compared. Results: The content of crude protein in powder slices of CCP with blood was higher than that of CCP with blood (P 0.05). The crude protein content in wax slices of CCP with freeze-drying processing was less than that with boiling processing (P 0.05). The content of crude protein and TAA in wax slices of CCP is both significantly higher than that in powder slices, bee slices (P 0.05). Conclusio:n The difference is existed in content of crude protein and amino acids in CCP with different processing methods. The wax silices of CCP are significantly higher than that of powder slices and bee slices. And the difference in powder slices and bee slices is not significantly. The distribution of crude protein and TAA in different parts of CCP with freeze-drying processing is more uniform than CCP with boiling processing.
ABSTRACT
OBJECTIVE:To determine the content of urea in Urea [13C] capsules by high performance cation-exchange chroma-tography (HPCEC). METHODS:The determination was performed on Zorbax 300 SCX column with mobile phases consisting of acetonitrile-0.1% phosphoric acid (20:80,V/V) at the flow rate of 1.0 mL/min. The detection wavelength was set at 200 nm and column temperature was 35 ℃. The sample size was 20 μL. RESULTS:The linear range of urea was 0.0039-1.0030 mg/ml(r=0.9997). The limit of quantitation was 3.918 μg/mL and the limit of detection was 0.975 μg/mL. RSDs of precision,stability and repetitive test were all lower than 2.0%. The recovery ranged 99.3%-101.0%(RSD=0.67%,n=9). CONCLUSIONS:The meth-od is simple,rapid,sensitive and suitable for the content determination of urea in Urea [13C] capsules.
ABSTRACT
OBJECTIVE:To establish a method of contents determination of 17 amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang. METHODS:Cation-exchange column was used to separate 17 kinds of amino acids;post-column derivatiza-tion liquid chromatography was used to determine the contents of amino acids:the column was strong sulfonic acid cation exchange resin LCAK06/Na with mobile phase A(weighing trisodium citrate 11.9 g,citric acid 6 g,phenol 1 g,dissolving by water,then adding 65 mL of methanol and 6 mL of concentrated hydrochloric acid,bringing the volume with tap water,adjusting pH to 3.4), mobile phase B(weighing trisodium citrate 11.9 g,NaOH 2.8 g,phenol 1 g,boric acid 5.0 g,adding water to dissolve,adjustingpH to 10.8),gradient elution,flow rate was 0.45 mL/min for A pump and 0.25 mL/min for M pump,the detection wavelength was 440 nm for proline and 570 nm for the remaining amino acids,the injection volume was 50 μL. RESULTS:The linear range were 20~400 nmol/mL of aspartic acid,threonine,serine,glutamic acid,glycine,alanine,valine,methionine,isoleucine,leucine,ty-rosine,phenylalanine,histidine,lysine,arginine,proline,10-200 nmol/mL of cystine(r were higher than 0.9890);RSDs of preci-sion,stability and reproducibility tests were lower than 2.0%;limits of detection were 0.16 nmol/mL except for cystine(0.08 nmol/mL);recovery was 98.5%-99.5%(RSD was 0.06%-0.21%,n=6). CONCLUSIONS:The method is simple with good precision, stability and reproducibility,and can be used for the simultaneous determination of amino acids in Fritillariae Pallidiflorae Bulbus produced in Xinjiang.
ABSTRACT
Objective:To compare the content differences of 18 amino acids in plancenta histolysate determined by pre-column de-rivatization HPLC and post-column derivatization cation-exchange chromatography ( AARO) . Methods: The HPLC method was per-formed on a C18 column and 2, 4-dinitro chlorobenzene ( DNCB) was used for pre-column derivatization, and then the determination was carried out after adding 0. 1 mol·L-1 borax buffer (pH=9. 1), and the AARO method was used for the direct determination with a strong acid cation-exchange chromatographic column and post-column derivatization. Results: The RSD for reproducibility of the AARO method was 1. 84%-0. 91%, while that of the HPLC method was 1. 87%-1. 04%. Conclusion:Both AARO method and HPLC method can be used for the quantitative determination of 18 amino acids in plancenta histolysate with the similar results. However, pre-column derivatization HPLC method may cause incomplete derivatization and instable derivatives.
ABSTRACT
Objective To construct a prokaryotic expression vector pET-22b+with Middle East respiratory syndrome ( MERS) coronavirus nuclocapsid protein( NP) gene and to express and purify N protein.Methods N gene amplified by PCR was inserted into the prokaryotic expression vector pET-22b+.Recombinant plasmid was confirmed using DNA elec-trophoresis and sequencing.NP was expressed in E.coli BL21(DE3) by IPTG induced and purified by cation exchange chromatography using the AKTA purification system.Results The NP gene sequence was proved to be correct by sequen-cing and the protein was expressed in both soluble and insoluble forms in E.coli BL21 ( DE3 ) after IPTG induction.The purity and concentration of recombinant protein was improved obviously by cation exchange chromatography and enrich-ment.Conclusion Recombiant NP of high purity and concentration is purified and will facilitate NP functional research.
ABSTRACT
Two methods of purification of the plantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31 are used in this study - the method of ammonium sulfate precipitation, Sep-pack C18 cartridge and reverse-phase HPLC chromatography on C18 Nucleosil column, and the method of direct purification by cation exchange SP Sepharose Fast Flow column Amersham (Pharmacia Biotech). The purity of the products from the two experimental protocols are examined for their molecular weight, aminoacid composition and sequence. Comparison of results show that the plantaricins purified with the two methods are identical. Both methods may be used to purify plantaricin ST31. Comparison of the yield in the purification protocols is 0.8% in the HPLC experimental protocol and 5.9% in the cation-exchange chromatography method.
Dois métodos de purificação de plantaricin ST31, uma bacteriocina produzida por Lactobacillus plantarum ST31 foram usados neste estudo - o método de precipitação pelo sulfato de amônia usando cartucho Sep-pack C18 para a filtração e HPLC de fase reversa em coluna de C18 Nucleosil, e o método de purificação direta por troca catiônica SP Sepharose "Fast Flow column Amersham" (Pharmacia Biotech). A pureza dos produtos obtidos pelos dois protocolos foi examinada através da determinação dos pesos moleculares, composição e seqüência dos aminoácidos. A comparação destes resultados revelou que, em termos da pureza dos produtos, não havia diferenças entre os dois métodos de purificação podendo-se, portanto, utilizar qualquer um dos protocolos de purificação testados. No entanto, o rendimento da purificação pelo método da troca catiônica foi de 5.9% enquanto o do método HPLC foi de 0.8%.
ABSTRACT
Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.
ABSTRACT
A 36-year-old pregnant woman with gestational diabetes mellitus and anemia was found to have an abnormal Hb (comprising 18.7%) in the automated midget low pressure cation- exchange chromatography (DiaSTATTM, Bio-Rad, USA) for Hb A1c assay. The abnormal Hb revealed an abnormal peak emerged slightly later than normal Hb A1 in DiaSTATTM chromatogram, subsequently confirmed by cellulose acetate membrane electrophoresis and isoelectric focusing. This hemoglobinopathy with high isoelectric point was noted and abnormal chain globin was prepared by chromatography. Family study was carried out and this chain variant was also found in four other family members, and all of them had no clinical abnormalities, except well controlled diabetes. As the results from peptide mapping, amino acid analysis and sequencing, abnormal Hb of the patient was finally identified as Hb Queens[ 34 (B15)Leu-->Arg] without clinical abnormalities.