Effect of the Buffer Concentration on the Separation of Lactate Dehydrogenase Isoenzymes from Different Sources by Electrophoresis

Abstract

The separation of lactate dehydrogenase isoenzymes by zone electrophoresis using cellulose acetate strips as support was dependent on the concentration of the buffer used (5 mM and 50 mM, pH 7.4) and on the source of the material (chicken liver or guinea-pig liver).

In three different 5 mM buffer systems, pH 7.4 (phosphate, veronal and Tris-HC1) the four lactate dehydrogenase isoenzymes present in chicken liver cytosol: M₃H, M₂H₂, MH₃ and H₄ were resolved into four separated bands. M₃H and M₂H₂ isoenzymes migrated towards the cathode whereas the other two isoenzymes showed anodic mobilities. In 50 mM buffers, pH 7.4 all enzyme activity appeared as a single band with anodic mobility similar to that of H₄. Guinea-pig liver isoenzymes were well resolved in both buffer conditions and appeared as five bands with anodic mobilities.

The different behaviour of the lactate dehydrogenase isoenzymes in 5 mM and 50 mM buffers can not be assigned to ionic strength effects but it may explained by assuming the binding of buffer anions to the different isoenzymes. The binding would increase with the molar concentration of the buffer and reduce charge differences among the isoenzymes to different extents depending on the source of the enzyme, chicken or guinea-pig liver.     

Rapid analysis of bovine milk proteins by fast protein liquid chromatography

The separation of bovine milk proteins by fast protein liquid chromatography has been investigated by ion-exchange chromatography on Mono Q and Mono S columns and by gel filtration on a column of Superose 12. The four major casein components (alpha s1, alpha s2, beta and kappa) as well as the minor gamma-caseins were generally well separated on the Mono S column with urea-containing buffers at pH 3.8 in as short a time as 7 min, although there was considerable overlap between alpha s1- and alpha s2-casein peaks. Peak area measurements indicated that the four caseins alpha s1, alpha s2, beta and kappa were present in total casein in the approximate proportions of 3.0:0.5:3.4:0.9, in good agreement with other literature values. Whey proteins were not separated on the Mono S column, but were all well resolved by rapid analysis on the Mono Q column at pH values between 6 and 8 in buffers free of urea or 2-mercaptoethanol. Both urea and 2-mercaptoethanol were required for casein analyses on the Mono Q column, but all the casein components were then separable over a broad pH range (5.0-11.0). While urea levels of 4.5-8.0 M and pH values of 7.0 to 8.0 were most generally useful, the resolution of some components was affected by urea concentration or pH, so conditions may have to be modified for specific analysis problems. The caseins were too similar in size to be separated on the Superose 12 column but high-speed gel filtration in as little as 15 min separated all the whey proteins well, molecular weight values obtained being in good agreement with literature values.     

Anion-exchange fast protein liquid chromatographic characterization and purification of apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E from human plasma

This paper describes a procedure for the rapid isolation of urea-soluble apolipoproteins (apo) from delipidated human very-low- and high-density lipoproteins using anion-exchange fast protein liquid chromatography. The separation was complete within 30 min and peaks corresponding to apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E were identified by comparing their chromatographic, electrophoretic and immunological behaviour with that of purified standards of each protein. A second purification step is necessary to obtain pure apolipoproteins. Apo E, which is difficult to purify by conventional chromatography, has been obtained in a good yield. The apo C-II that was obtained produced a symmetrical peak on chromatography but three bands in isoelectric focusing. The method can be upgraded to a preparative scale and offers the possibility of direct purification of apolipoproteins both from high-density lipoproteins and (following preliminary gel chromatography) from very-low-density lipoproteins.     

Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis

A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.     

The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7-10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti-human IgG followed by an enzyme-labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.     

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.     

Separation and recovery of nucleic acids with improved biological activity by acid‐degradable polyacrylamide gel electrophoresis

One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid‐degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44–84% of DNA. The second conventional (non‐degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid‐degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid‐degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid‐degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离

建立苯胺甲基键合硅胶固定相(PAMS)高效液相色谱分离核苷与碱基的方法；研究流动相有机溶剂浓度、磷酸缓冲液pH值、离子强度对核苷和碱基在该键合固定相上的色谱保留及分离选择性的影响，用磷酸缓冲液(pH＝4)为流动相快速分离了部分核苷与碱基。     

Isotachophoresis of proteins

The analytical separation of proteins by isotachophoresis (ITP) was achieved in a short electrophoretic path and with a resolution comparable to that of isoelectric focusing by the appropriate selection of (1) a mixture of ampholytes as spacers to generate linear gradients of electrophoretic mobility and (2) the counter ions chosen to buffer the complete pH gradient generated. This ITP technique is exemplified by the analysis of plasma proteins in agarose gels. Up to 46 samples in the same gel plate were analysed. The resolution was such that at least 30 clear and discrete bands per sample could be observed after staining with Coomassie Brilliant Blue. The resolving power of ITP could be further increased for the study of a particular protein or zone by the selection of suitable spacers and counter ions.     

alpha-N-benzoylarginine-2-naphthylamide hydrolase (cathepsin B1 ?) from rat skin. II. Purification of the enzyme and demonstration of two inhibitors in the skin.

A thiol-activated, alpha-N-benzoylarginine-2-naph-thylamide (BANA) hydrolyzing enzyme was purified from rat skin by ammonium sulfate precipitation, gel filtration, and DEAE cellulose chromatography. In DEAE cellulose chromatography the enzyme was fractionated into two multiple forms (preparations I and II). The activity of undiluted preparation II, but not that of preparation I, was increased, when the enzyme was preincubated at pH 4, at 55 degrees C. Simultaneously, the isoelectric point of preparation II was shifted to that of preparation I, i.e., from 6.2 to 7.5. Activated preparation II behaved in DEAE cellulose chromatography as preparation I. Molecular weights of the enzymes of both preparations were 27 000, and pH optima were at pH 5.8 and 7.0, for BANA and leucine-2-napthylamide (Leu-NA), respectively. The BANA and Leu-NA hydrolyzing enzymes could not be separated by gel filtration, DEAE, CM, or Amberlite IRC-50 chromatography, isoelectric focusing, or analytical polyacrylamide gel electrophoresis. Two inhibitors of BANA hydrolase were demonstrated by gel filtration in the salt precipitated skin extract. The activities of the BANA hydrolase preparations did not increase linearly with increasing enzyme concentration, with the exceptions of activities of preparation I and acid-activated preparation II. The role of the inhibitors in the nonlinearity of the activity/enzyme concentration curves is discussed.     

Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence combined with gel filtration chromatography and isoelectric focusing separation

Synchrotron radiation X-ray fluorescence (SRXRF) spectroscopy is an advanced method of quantitative multielemental analysis with space resolution of several microm and sensitivities in the microg g(-1) range. It can be used for keeping track of trace elements after an electrophoretic separation of biological samples. In this paper, proteins in human liver cytosol were separated with gel filtration chromatography and thin layer isoelectric focusing (IEF). The contents of metal ions in protein bands were determined by SRXRF. The results showed that in the molecular weight (MW) range of 10 approximately 25 kDa, there were at least 2 Zn-containing bands with isoelectric point (pI) of 5 approximately 6 and 6.2 approximately 7, respectively and about 11 Fe-containing proteins with pI of 4.4, 4.6, 4.8, 5.0, 5.2, 5.3, 5.5, 5.6, 6.6, 6.8, and 7.2, respectively, present in human liver cytosol. The Zn-containing band with pI of 5-6 is the dominant species of zinc in this MW range. In addition, the Cu-containing bands with pI of 5.0 and below 4.8 were also detected. It is demonstrated that the procedure could be widely used in further investigations of the chemical species of trace elements in biological samples.     

Solubilization and purification of galactosyltransferase from golgi membranes of rat ventral prostate

UDP-galactose ovomucoid galactosyltransferase, a membrane-bound enzyme involved in the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences on glycoproteins, has been solubilized and purified from rat ventral prostate Golgi membranes. Solubilization was effected by treatment of the particulate fraction with Triton X-100 (0.5% v/v) and MnCl₂ (25 mM). The solubilized enzyme was purified by affinity chromatography on hen ovomucoid-sepharose column. The purified galactosyltransferase showed three protein bands of approx. 74,000, 60,000, and 54,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity eluted at approx. 70,000 daltons with a broad shoulder between 60,000 and 50,000 daltons. Isoelectric focusing of the purified enzyme resolved at least five active bands with pHi of 9, 7.4, 6.75, 6.1, and 4.8.     

Purification of porcine proinsulin by high-performance liquid chromatography

A procedure has been developed for purification of porcine proinsulin by high-performance liquid chromatography from a preparation obtained as a side product during the Sephadex G-50 gel filtration of an impure porcine insulin preparation. Reversed-phase chromatography was carried out on octadecylsilica as the stationary phase with graded mixtures of acetonitrile or methanol-acetonitrile and phosphate buffer pH 2.4 as the mobile phase. The crude preparation separated into five different groups of proteins, the proinsulin-containing peak being identified by the co-eluting internal proinsulin marker. After purification by conventional procedures (separation, pooling, freeze drying, desalting, reprecipitation and drying) this peak fraction was rechromatographed by high-performance liquid chromatography (for final purification) to give a single peak protein which had identical electrophoretic mobility to that of commercial porcine proinsulin, and which converted to a protein with electrophoretic mobility similar to that of porcine insulin.     

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS‐PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA‐F8 (anti‐MHC slow/I), SC‐71 (anti‐MHC 2a and 2x), 10F5 (anti‐MHC 2b), and BF‐35 (anti‐MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS‐PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS‐PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.     

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract

The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.     

Purification of cytochromes P-450 derived from liver microsomes of untreated and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated marmoset monkeys

The purification of multiple forms of cytochrome P-450 (P450) from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated marmosets using fast protein liquid chromatography (FPLC) is described. The main aim was to achieve a better separation of certain closely related P450 sub-forms from each other than that previously obtained using conventional chromatography. An 8-aminooctyl-Sepharose fraction of cholate-solubilized microsomes was obtained first and, after fast desalting on Sephadex G-25, loaded on to a preparative Mono Q column. Five of the six gradient peaks contained P450 and were each rechromatographed on an analytical Mono Q column. The pass-through peak was fractionated further using a Mono S column. Other HPLC-quality anion- and cation-exchange gels were compared. For removal of excess of non-ionic detergent, five types of hydroxyapatite gels were compared. Seven purified forms of P450 and cytochrome b5 and P420 were isolated and characterized according to PHAST sodium dodecylsulphate-polyacrylamide gel electrophoretic apparent molecular masses, catalytic, spectral and magnetic properties and also TCDD-binding capacity (molar ratio of [14C]TCDD to P450). There are at least two sub-forms which appear to be TCDD inducible, one showing a substantial ethoxyresorufin-O-deethylase activity and the other having a high TCDD-binding molar ratio. Two other forms appear to be constitutive, as deduced from comparisons with forms purified from untreated animals.     

Chemical Basis of the Action of Glyoxalase I,an Anticancer Target Enzyme

Although glyoxalase I was discovered in 1913 the physiological role of this ubiquitous enzyme is still far from clear. It catalyzes the reaction of α-ketoaldehydes and glutathione to produce S-D-lactoylglutathione, from which D-lactate and glutathione are produced by glyoxalase II. Argument raged for many decades about the nature of the natural substrate. Was it methylglyoxal? Was methylglyoxal ever formed metabolically or was it purely artifactual? Some of these questions have been resolved in that a number of metabolic processes which produce α-ketoaldehydes have now been recognized. Equally unsuccessful have been attempts to ascribe a physiological role to glyoxalase. This is clearly an important question since glyoxalase I occurs in cells at all levels of evolution. Time and time again glyoxalase I has been claimed to be linked to cancer, and a number of research groups worldwide are using it as a model for designing potential anticancer drugs. In this review article the mechanism of action of the enzyme is discussed, knowledge of which enables stronger inhibitors to be synthesized. Until roughly ten years ago when powerful NMR techniques were used to study it for the first time, it was assumed that the key step in the mechanism was a hydride ion transfer. Today, the mechanism is envisaged as a proton transfer.     

Capillary zone electrophoresis and micellar electrokinetic chromatography of solution of polyaniline particles

The behavior of two different fractions of the electrically conducting polymer polyaniline (PANI) was studied in order to characterize them using fast, simple, and cheap methods of capillary zone electrophoresis. In contradistinction to the expectations that the particles will bear a positive charge on their surface, in the milieu of electrolytes used for electrophoresis (0.01-0.05 M buffers of pH 4-8.5), the surface was demonstrated to be slightly negatively charged, which led to a slow migration to the anode. An evident charge was given to the particles after their interaction with micelles formed from surface active components, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). An active electrophoretic migration and full separation of the PANI fractions was achieved by micellar electrokinetic chromatography (MEKC) with negatively charged SDS micelles. With this method, a reliable qualitative as well as quantitative analysis was performed in the concentration range of 0.1-1.6 mg/mL with a reproducibility of migration times better than 1.5%.     

Major histocompatibility complex (MHC) protein analysis by optimised two-dimensional electrophoretic methods

Human histocompatibility molecules HLA-Class I and Class II (DR, DQ, DP) were analysed using three two-dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing-acidic gradient (IEF-AG) and isoelectric focusing-basic gradient (IEF-BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrofocusing profiles. The NEPHGE protocol was adequate for separating proteins across a broad range of pI mobilities, i.e. 4.4 pH units between the acidic and the basic end. In contrast, the IEF-AG and the IEF-BG protocols gave a separation power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF-AG and -BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF-BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA-DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing.