Rapid separation of anionic oligosaccharide species by high performance liquid chromatography

We have developed a method for the rapid separation of anionic oligosaccharide species by high-performance liquid chromatography utilizing a MicroPak AX-10 ion-exchange column (Varian Associates) with the mobile phase consisting of 25–500 mm KH₂PO₄, pH4.0. Separation of oligosaccharides bearing zero, one, two, three or four sialic acid residues requires less than 45 min. Oligosaccharides containing mannose-6-PO₄ moieties in monoester or diester linkage can also be analyzed in this system. Preparative separations of as much as 20 mg of oligosaccharide can be accomplished in a single chromatographic analysis with quantitative yields of oligosaccharide. This method should prove useful for the rapid isolation and characterization of anionic oligosaccharide species.     

Characterization of a new substrate for protein kinase C: assay by continuous fluorometric monitoring and high performance liquid chromatography

A synthetic peptide derived from the phosphorylation site in the beta-subunit of phosphorylase kinase (RTKRSGSVYEPLKI) is an efficient substrate for rat brain protein kinase C: Km = 18 +/- 2 microM and Vmax = 2.1 +/- 0.1 mumol/min/mg. The phosphorylation of the peptide, which occurs at Ser7, can be followed by four independent procedures. 1. Standard measurement of 32P incorporation. 2. Reverse phase HPLC in a gradient system containing 0.1 M ammonium sulfate in the stationary phase. 3. Continuous fluorometric monitoring of the changes in intrinsic peptide fluorescence. 4. Continuous fluorometric determination of NADH oxidation in a coupled enzyme assay.     

Rapid purification of RNAs using fast performance liquid chromatography (FPLC)

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.     

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.     

Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1,500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

Rapid purification of bacterially expressed fusion proteins by high-performance liquid chromatography methods

Recent developments in recombinant DNA technology allow the high-level expression in bacteria of substantial amounts of viral and eukaryotic proteins whose genes have been cloned into plasmids. The present study reports two high-performance liquid chromatography (HPLC) methods for the rapid purification to apparent homogeneity of these bacterially expressed proteins. The two methods are anion exchange HPLC in the presence of 7 M Urea and reverse-phase HPLC of protein solubilized by 7.0 M guanidine hydrochloride. The two methods have been used successfully to purify fusion products of the v-myb oncogene and fusion proteins from HTLV-I Px and transmembrane regions and should be of general utility for purification of other bacterially produced proteins.     

Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography

Unquestionably, the purification of polypeptides by chromatographic methods is a considerable bottleneck in their preparation. Peptides synthesised by solid phase synthesis typically contain chromatographically similar impurities that complicate purification by reversed phase high performance liquid chromatography (HPLC) techniques. We report on the application of a slow gradient HPLC protocol that allows, in a single chromatographic step, the purification of hundreds of milligrammes of material. This technique was applied to an extensive collection of synthetic polypeptides some incorporating non‐proteinogenic functionality. In all cases examined, the peptides were not only obtained in high purity peptides but were also recovered in multi‐milligramme amounts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Rapid purification of a high molecular weight human brain protein by chromatography on controlled pore glass.

Controlled pore glass (CPG) chromatography was employed to simply (one pass through column) and rapidly (60 minutes) purify a human brain specific protein having a high molecular weight (approximately 250,000 mol. wt.) from a crude brain extract containing proteins of varying molecular weights. This method, either exactly as described herein or by adjusting the pore size of the CPG, should be adaptable to other purification problems.     

Rapid analysis and purification of polymerase chain reaction products by high-performance liquid chromatography

This report describes the use of high-performance liquid chromatography (HPLC) for the rapid analysis and purification of the polymerase chain reaction products. Employing a new anion-exchange nonporous column, efficient separations of both DNA restriction fragments and amplified PCR products are achieved in 10 to 20 minutes and quantitated within +/- 10%. The performance of the HPLC technique is described in terms of resolution, reproductibility, sensitivity and micropreparative capability and compared to that of gel electrophoresis for this application.     

Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments

A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogeneous after purification and exhibits a molecular mass of approximately 85 kDa, as determined by SDS/PAGE. From its chromatographic behaviour on hydroxyapatite, and from Western-blotting experiments using isoenzyme-specific antibodies, we were able to identify the bovine rod outer segment protein kinase C as being of the alpha or type-III form. The purified protein kinase C has a specific activity of 1066 nmol 32P.min-1.mg protein-1, and shows a 30-fold activation upon the addition of the effectors Ca2+, PtdSer and 1,2-diacylglycerol. Arachidonic acid and linoleic acid were also found to enhance significantly the activity of the purified enzyme.     

Analysis of bacterial menaquinone mixtures by high performance liquid chromatography

Menaquinone mixtures of microbial origin were separated according to the chain length and the degree of hydrogenation of the polyisoprenyl side-chain by reversephase partition high performance liquid chromatography. Menaquinones can be measured easily and sensitively by the chromatographic system described here.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.