Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Ion-exchange high-performance liquid chromatography of oligodeoxyribonucleotides using formamide

The superiority of buffer systems containing formamide for the ion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotide mixtures generated in solid-phase syntheses is illustrated. The resolutions achieved are compared to those achieved with the same mixtures in other eluting solvents. The use of formamide systems is recommended for oligodeoxyribonucleotide purification in general and is particularly valuable where the oligonucleotide of interest is highly self-complementary and/or rich in deoxyguanosine residues.     

Determination of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) activity by high-performance liquid chromatography and electrochemical detection

The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.     

Teleonomical optimization of a fractal model of the pulmonary arterial bed

Modeling the pulmonary arterial tree (PAT) is considered here as an optimal synthesis problem. Firstly, a class of candidate models is specified: the three-dimensional symmetric dichotomous fractal trees of elastic tubes described by Womersley's equations. Secondly, the parameters are shown to be constrained by interactions of PAT with the rest of the body; these constraints are used to limit the volume of the parametric space to which attention will be directed in the synthesis step. Thirdly, a teleonomical hypothesis is proposed: a naturally selected PAT must have a minimal input impedance under conditions keeping total arterial volume and distensibility as small as possible. This hypothesis is translated in mathematical terms and the resulting cost-function minimized in the limited parametric volume. The optimal model has parameter values and an impedance spectrum corresponding satisfactorily with real data. Moreover this model gives a clear picture of the internal hemodynamic behavior of PAT as an impedance matching device.     

Automated, High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Automated,High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Assay of galactosyltransferase by high-performance liquid chromatography

Galactosyltransferase catalyzes transfer of galactose from UDP-galactose to glucose or N-acetylglucosamine with resultant formation of galactosides and UDP. In this new assay galactosyltransferase activity is measured by determining UDP by isocratic high-performance liquid chromatography on an amino-bonded column monitored spectrophotometrically. Concurrently, unreacted UDP-galactose and breakdown products arising from UDP-galactose (UMP and uridine) are also determined. The new technique does not require radioactive substrates, permits usage of saturating concentrations of UDP-galactose, and provides monitoring of side reactions.     

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S-carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.     

Degradation of luteinizing hormone-releasing hormone by serum and plasma in vitro

Luteinizing hormone-releasing hormone (LH-RH) is degraded in vitro by serum and plasma from several species (human, rat, guinea-pig and cattle). Separation of the degradation products by high-performance liquid chromatography (HPLC) followed by amino acid analysis and radioimmunoassay showed that the main sites of cleavage are the Trp₃-Ser₄ and Tyr₅-Gly₆ bonds. Two peptidases are responsible since the cleavage at Trp₃-Ser₄ can be selectively inhibited by EDTA. In human plasma, the peptidase responsible for Trp₃-Ser₄ hydrolyssishas a K_m of 2.9 · 10^?4 M and V of 30 nmol/h per ml plasma. The half-life in vitro of LH-RH in serum and plasma from various species ranges from 3 h (guinea-pig) to 9.8 h (human). The peptidase cleaving LH-RH at Tyr₅-Gly₆ is present as an impurity in some commercial bovine serum and plasma albumins. Such contamination may have important practical implications for work involving peptide assays where albumins are used as carrier proteins.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-performance liquid chromatography for assaying NAD glycohydrolase from Neurospora crassa conidia

A rapid and sensitive high-performance liquid chromatographic technique was developed to determinate NAD glycohydrolase (EC 3.2.2.5.) activity from Neurospora crassa conidia. The separation of the assay substrate and products was achieved by isocratic reverse-phase chromatography and the peaks were detected by the absorbance at 259 nm. Quantities of NAD+ and nicotinamide as small as 10 pmol could be measured.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

Identification of eleven human hemoglobin variants by high-performance liquid chromatography: Additional data on functional properties and clinical expression

Eleven abnormal hemoglobins were detected in the course of cord blood screening or in the evaluation of evident hematological problems in individual cases. Identification of the variant in each case was done by high-performance liquid chromatography (HPLC); HPLC provides a rapid, sensitive means for the examination of abnormal hemoglobins. Some of the 11 variants that were identified have been described repeatedly and are included to provide information on the HPLC behavior of tryptic peptides. Others are much rarer. Additional information is provided about the hematological and clinical expression as well as ethnic and geographical distribution of the abnormal hemoglobin.This investigation was supported in part by Grants HL-02558 and HL-15162 from the National Institutes of Health, U.S. Public Health Service.     

魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

Hierarchical gene synthesis using DNA microchip oligonucleotides

High-cost of oligonucleotides is one of the major problems to low-cost gene synthesis. Although DNA oligonucleotides from cleavable DNA microchips has been adopted for the low-cost gene synthesis, construction of DNA molecules larger than 1 kb has been largely hampered due to the difficulties of DNA assembly associated with the negligible quantity of chip oligonucleotides. Here we report a hierarchical method for the synthesis of large genes using oligonucleotides from programmable DNA microchips. Using this hierarchical method, we successfully synthesized 1056 bp Dpo4 and 2325 bp Pfu DNA polymerase genes as models. This hierarchical strategy can be further expanded for the syntheses of multiple large genes in a scalable manner.     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.     

High-performance liquid chromatography of fatty acid isopropylidene hydrazides and its application in lipid analysis

Fatty acid isopropylidene hydrazides, prepared by stepwise treatment of acyl lipids with hydrazine and acetone, were analyzed by high-performance liquid chromatography on a reversed-phase column. These derivatives could be easily eluted with 15% water in methanol and monitored by measuring absorbance at 229 nm with a uv detector. Their elution behavior, in general, was similar to that of methyl esters and some commonly used ultraviolet-absorbing derivatives of fatty acids. The new method has been used for fatty acid analysis of some oils.