Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

A range of high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm have been constructed by securing glass or plastic tubing into standard HPLC fittings. These were packed with chromatographic materials chosen for operation at moderate pressures with high flow rates. These columns were shown to be effective in a conventional HPLC instrument for peptide and protein separations in reverse-phase mode and for proteins in ion-exchange and size-exclusion modes. The simple construction and low cost of these microbore columns allow them to be considered as disposable. Using only small amounts of any type of packing material, they have the flexibility to be adapted to a wide range of analytical and micropreparative separations.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide and secretin from the ovine small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucinamide (PHI) and secretin were separated and purified to homogeneity from ovine small intestine, using radioimmunoassay and radioreceptor assay for detection. An efficient and rapid purification sequence included acid extraction, concentration on a bulk C18 cartridge, filtration on a Fractogel column, ion-exchange chromatography on Mono-S and a maximum of three successive reverse-phase HPLC steps. The amounts of peptides obtained from 450 g wet weight tissue were 20 micrograms VIP, 15 micrograms PHI and 5 micrograms secretin. The as yet unknown amino acid sequences of the three peptides were found to be identical to those of the corresponding bovine peptides.     

Glicentin is present in the pig pancreas

Specimens from porcine pancreas and ileal mucosa were extracted in acid/ethanol, subjected to gel permeation chromatography, ion-exchange chromatography, enzymatic peptide degradation, reverse-phase HPLC, and analysed for glucagon-like and glicentin-like immunoreactivity by region-specific radioimmunoassays. Results obtained with all methods were consistent with the hypothesis that glicentin is present in the pig pancreas in small amounts.     

Purification of human epidermal growth factor (urogastrone) from urine

Human epidermal growth factor has been isolated from a concentrated chromatographic eluate of human urine. The purification method utilizes six chromatographic steps including adsorption to aminoethylcellulose (AE-11), gel filtration on Sephadex G-50, carboxymethylcellulose (CM-52) chromatography, ion-exchange HPLC and reverse-phase HPLC. The final product appears homogeneous and identical to pure gamma-urogastrone when analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC using two eluent systems. The yield of the method described above allowed the development of a sensitive radioimmunoassay system for this growth factor.     

Quantitative HPLC analysis of acidic opines by phenylthiocarbamyl derivatization

A new method for the quantitative analysis of acidic opines--alanopine, strombine, tauropine, and beta-alanopine--is presented. The method is based on formation of phenylthiocarbamyl (PTC) derivatives of the acidic opines after partial purification by ion-exchange chromatography. The PTC acidic opines are separated by reverse-phase high-performance liquid chromatography (HPLC) and detected within 20 min by ultraviolet absorbance. This HPLC method gives higher sensitivity, 10-30 pmol minimum detection, and better reproducibility than the high-voltage paper electrophoresis method. There is also good agreement for the three acidic opines (alanopine, strombine, and tauropine) when compared by HPLC and electrophoresis methods. Accumulation of beta-alanopine was observed for the first time in the adductor muscle of blood shell, Scapharca broughtonii, during aerial exposure by application of the HPLC detection method.     

Amino acid sequence of a soybean (Glycine max) seed polypeptide having a poly(L-aspartic acid) structure

A polypeptide of Mr 4400 was isolated from soybean (Glycine max) seeds by extraction with 60% ethanol followed by ion-exchange and reverse-phase chromatography. The peptide contains an unusually high amount of aspartic acid residues (approximately 25%, and hence is designated as soybean aspartic acid-rich peptide). Its complete primary structure was determined by conventional methods to be the following. Ser-Lys-Trp-Gln-His-Gln-Gln-Asp-Ser-Cys-Arg-Lys-Gln-Leu-Gln-Gly-Val-Asn- Leu-Thr-Pro-Cys-Glu-Lys-His-Ile-Met-Glu-Lys-Ile-Gln-Gly-Arg-Gly-Asp-Asp- Asp-Asp-Asp-Asp-Asp-Asp-Asp A striking features of this primary structure is the presence of a poly(L-aspartic acid) sequence at the carboxyl terminus, and this polyaspartyl segment was found to precipitate bovine trypsin. The presence of the putative cell attachment sequence Arg-Gly-Asp was also noted.     

High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide.

This paper reports a high-performance liquid chromatographic (HPLC) technique to determine biotin in biological samples. Biotin and the internal standard dethiobiotin are converted into fluorescent derivatives by using panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] as a fluorescence label. Biotin is extracted from biological tissue with trichloroacetic acid and the extract is purified by a combination of solid-phase extraction on C18 cartridges, ion-exchange chromatography on DOWEX formate resin, and thin-layer chromatography. The purified sample extract is derivatized in the presence of a crown ether. The resulting panacyl esters can be separated on reversed-phase as well as on normal-phase HPLC. Normal phase HPLC is preferable because it provides higher sensitivity and demands less sample pretreatment. Analysis of rat intestinal tissue revealed that only about 13% of the biotin is present in free form whereas 87% is bound in proteins from which it can be released by hydrolysis. Biotin values determined by this method are comparable to those obtained by other techniques.     

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.     

Postcolumn detection of serum proteins with the biuret and Lowry reactions

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.     

Isolation of heavy chain isoenzymes of myosin subfragment 1 by high performance ion exchange chromatography

The procedure of high performance ion-exchange chromatography has been used to fractionate subfragment 1 of myosin (SF1) into its isoenzymic forms. In contrast to conventional ion-exchange procedures which yield two fractions corresponding to SF1(A1) and SF1(A2), the high performance liquid chromatography (HPLC) procedure resolves SF1 into four discrete fractions. The first pair that is eluted appears to be A1-containing isoenzymes while the latter pair corresponds to the A2 forms based on their polypeptide compositions by gel electrophoresis in the presence of sodium dodecyl sulfate. By gel electrophoresis under nondenaturing conditions it is not possible to differentiate between the two fractions corresponding to each isoenzyme. Although very minor differences between the fractions can be seen by the presence of extraneous peptides, these are present in far below stoichiometric amounts and, therefore, make it very unlikely that the superior fractionation by the HPLC procedure is based on their presence. An examination of the heavy chain heterogeneity in each of these fractions by peptide mapping revealed that the extra separation was based on this factor. Thus the HPLC procedure is capable of providing separation of SF1 into heavy chain-based isozymes as well as the light chain forms. ATPase measurements of these fractions reveal only minor differences in the Ca2+- and EDTA-activated ATPase.     

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity iodinated imidoester: preparation, characterization, and binding to isolated pancreatic acinar cells

Proteoglycans were separated by high-performance liquid chromatography (HPLC), using two coupled Aquapore columns containing glycerylpropylsilane groups covalently linked to large-pore (50–100 nm) silica spheres. This two-column HPLC system was effective in separating cartilage proteoglycan aggregates and monomers, without altering their biochemical integrity. This system was also effective in resolving small amounts of isotopically labeled proteoglycans synthesized by cultured mammalian cells. The small sample size, short analysis time, and high reproducibility represent improvements in the study of proteoglycans over conventional soft-gel chromatography.     

Glutamate receptor antagonists from the spider Argiope lobata venom

Homologous low molecular weight compounds blocking postsynaptic glutamate receptors were isolated from the Argiope lobata spider venom by ion-exchange chromatography and reverse-phase HPLC. Structures of nine different blocking agents were determined by NMR and mass spectroscopy. They can be divided into three groups: argiopin, argiopinins and pseudoargiopinines. The major principles of the structural organization of the novel class of antagonists of glutamate receptors were considered.     

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.     

Identification and characterization of the emetic effects of peptide YY

Emesis was noted following intravenous bolus injections into dogs of a chromatographic subfraction derived from porcine small intestinal tissue extracts. The active agent was isolated from this subfraction using sequential ion-exchange and reverse-phase HPLC and demonstrated to be the recently identified regulatory peptide PYY. The threshold dose for PYY-induced emesis in the dog is less than 120 pmol/kg. Emesis was sometimes seen following large IV bolus doses of neuropeptide Y (NPY), but none was seen following IV injection of pancreatic polypeptide (PP). Dogs prepared with discrete, bilateral lesions of the area postrema were refractory to a suprathreshold emetic dose of PYY. PYY is the most potent, circulating emetic peptide identified to date.     

小牛胸腺新胸腺活性因子(TAF-Ⅰ)的纯化和鉴定

以小牛胸腺为原料 ,通过匀浆、冻融、超滤、SephadexG 15凝胶过滤、DEAE SephadexA 2 5阴离子交换层析和反相高效液相色谱等步骤 ,分离得到了胸腺活性因子Ⅰ (thymusactivityfactorⅠ ,TAF Ⅰ ) .5 0 0g小牛胸腺组织中纯化得到了 0 92mg胸腺活性因子Ⅰ .经ESI MS质谱鉴定 ,结果显示其分子量为 6 18 8.氨基酸组成分析显示它由Ala、Gly、Glu、Gln、Lys、Ser 6种氨基酸残基组成 .体外生物学活性实验证明 ,TAF Ⅰ能显著促进人外周血淋巴细胞的E 玫瑰花结的形成率和促进小鼠脾淋巴细胞的分裂增殖     

Isolation and Characterization of Marine Brevibacillus sp. S-1 Collected from South China Sea and a Novel Antitumor Peptide Produced by the Strain

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.     

普通离子交换剂用于HPLC柱层析法快速分离眼镜王蛇毒

目的　为了经济快速分离眼镜王蛇(Ophiophagushannah,Oh)蛇毒中的毒素成分。　方法　用普通离子交换剂于高效液相色谱柱 (HPLC) TSKgel SP-Toyopearl 65 0 SF (4× 1 5 0 mm)层析法 ,实验取得最佳分离条件后 ,将蛇毒样品上柱后进行梯度洗脱 ,各洗脱峰收集后在 Cosmosil 5 C4-AR-3 0 0柱 (4 .6× 1 5 0 mm)上进行逆相 HPLC分析。非单峰组分再进行 HPLC凝胶过滤柱TSKgel Toyopearl HW-40 Fine(4× 2 5 0 mm)层析 ,层析峰组分再进行 HPLC逆相分析。　结果　眼镜王蛇毒经HPLC离子交换柱层析获得了 1 6个蛋白组分 ,其中有 5个组分经逆相 HPLC分析单一组分 ;另外的复合性组分再进行 HPLC凝胶过滤柱层析后又得到 5个单峰蛋白组分。　结论　HPLC离子交换柱层析对分离蛇毒蛋白很有实用价值 ,特别是蛇毒样品量少的情况下 (1 0 ug)也能较好分离。还具有分离时间短 (1 h左右 ) ,无须低温条件等优点。HPLC凝胶过滤柱层析可进一步使蛋白组分得到提纯