Characterization of two types of mitochondrial creatine kinase isolated from normal human cardiac muscle and brain tissue

Two types of mitochondrial creatine kinase (Mi-CK), sarcomeric (sMi-) and ubiquitous (uMi-)CKs, were isolated from normal human cardiac muscle and brain tissue, respectively, and their heterogeneity was characterized by means of isoelectric focusing (IEF). Octameric sMi-CK and uMi-CK were electrophoresed cathodic to cytoplasmic muscle-type creatine kinase isoenzyme (CK-MM) and dimeric Mi-CKs were found at the position of CK-MM on a cellulose acetate membrane. The electrophoretic mobilities of sMi-CK were similar to those of uMi-CK. Octameric sMi-CK was focused at pI 7.1-8.0 and dimeric forms at pI 6.55, 6.75, 6.85, and 6.95. New bands appearing at pI 6.65 and 6.75 after treatment of sMi-CK with carboxypeptidase B were found to be delysined forms. sMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of sMi-CK for creatine phosphate (PCr) was 1.19 +/-0.20 mmol/L (mean +/- standard error), the activation energy (Ea) was 108.3+/-1.2 kJ/ mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 79.6+/-1.9%. On the other hand, octameric uMi-CK was focused at pI 7.1-7.9 and the dimeric forms were focused at pI 6.6, 6.7, 6.8, 6.9, and 7.0. Delysined forms were focused around pI 6.3, 6.4, 6.8, and 6.9. uMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of uMi-CK for PCr was 1.07+/-0.03 mmol/L, Ea of uMi-CK was 110.0+/-0.9 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 90.3+/-0.4%. The sMi-CK and uMi-CK were hybridized and the hybrid Mi-CK appeared at pI 6.78, 6.98, and 7.1-7.95. The pIs of the hybrid Mi-CK were between those of sMi-CK and uMi-CK. As described above, sMi-CK and uMi-CK were slightly different from each other with respect to the pI and some enzyme characteristics.     

Heterogeneity of serum creatine kinase isoenzyme-MM in acute myocardial infarction

Isoelectric focusing of serum creatine kinase (CK;EC 2.7.3.2) reveals up to 14 CK-MM subbands following acute myocardial infarction (AMI). The "normal" subbands 1 (pI 6.91), 2 (pI 6.65) and 3 (pI 6.35) are faintly present in normal serum and the "abnormal" subbands c (pI 7.25), e (pI 6.85), g (pI 6.50), i (pI 6.28), j (pI 6.20) and k (pI 6.15) are prominently detected in sera with elevated CK. "Abnormal" subbands a (pI 7.55),b(pI7.35),d(pI7.05),f(pI6.72) and h(pI6.40) have only been detected in AMI. The "abnormal" subbands appear, and reach maximum intensity (together with CK-MM 1-3), 3-12 h after infarction, and become faint and anodally convert (as do CK-MM 1-3) within 36 h. Similar changes are detected by nonequilibrium pH gradient electrophoresis which combines CK-MM and CK-MB analysis. In vitro incubation of serum with 0.015 M 2-mercaptoethanol induces conversion of CK-MM 1, 2 and 3 to b and c, d and e, and f and g, respectively. Thus, the complexity of the patterns is explained by a secondary conversion of "normal" to "abnormal" subbands superimposed upon anodal conversion of CK-MM 1----3. The clinical significance of these findings is discussed.     

High-performance liquid chromatographic determination of creatine kinase activity influenced by methylglyoxal

Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N-terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation.     

New azo dyes as colored isoelectric point markers for isoelectric focusing in acidic pH region

Newly prepared azo compounds and several commercially available indicators were investigated for their applicability as colored isoelectric point (pI) markers for isoelectric focusing (IEF) in the acidic range below pH 5. The majority of compounds described here can serve as primary standards since their pI values were determined by UV-VIS spectrophotometry independently IEF and direct measurement with a pH electrode. Subjected to gel IEF they show narrow and well-observable zones of different colors. Finally, our work resulted in suggestion of a color ladder composed of pI markers covering the pH range from 1.5 to 4.7.     

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

纳升电喷雾毛细管液相色谱-串联质谱鉴定K562细胞株中混合蛋白质

用标准蛋白质混合物建立了一种适用于低丰度混合蛋白质及其异构体分离与鉴定的蛋白质组学方法。通过IPG胶条等电聚焦分离蛋白质,染色后进行混合胶内酶切,采用纳升电喷雾毛细管液相色谱一串联质谱“散弹法（shot-gun）”分析酶切产物,并进行数据库检索鉴定蛋白质。运用该方法从K562细胞株样品中鉴定出14种具有重要功能的蛋白质,部分蛋白质同时在多个条带中出现,可能是异构体。肽段及其碎片离子的平均质量偏差小于0．05U,综合得分大都远远超过有效值。该方法灵敏、准确度高、分辨率高、省时、便于操椎存苍宗罾白甩异构体青而右优势．     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Isoelectric focusing in soil science

Applications of isoelectric focusing (IEF) in soil science are illustrated. The materials studied, i. e. humic substances and organic fertilisers, and their behaviour when subjected to IEF are briefly described. Preliminary studies carried out on soil indicate a relation between the isoelectric point and molecular weight, and a connection with humification has been hypothesised; therefore, assessment of soil conditions could be an application of IEF. Another application is evaluation of the stabilisation of organic fertilisers. Finally, IEF can be used to distinguish different organic matrices, as such and in mixtures with mineral fertilisers.     

Bioluminescence flow sensor for determination of creatine kinase activity in blood

A bioluminescent flow sensor was developed for the assay of creatine kinase (CK) using firefly luciferase immobilized on a nylon coil. The CK-catalysed reaction of creatine phosphate with ADP took place in a cuvette before the injection into the bioluminescent detector coil. The response was linear from 0.1 to 100 U l^? at 25°C. An advantage of the flow sensor is a detection limit of less than 0.1 U l^?1, which, together with a high precision, allows determination of the CK activity in blood sera in about 5 min. The intra- and inter-assay reproducibilities (RSD) were less than 10% and the recovery range was 86–110%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96-5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water-soluble fish muscle proteins on Servalyt Precotes 3-6.     

Two-dimensional gel isoelectric focusing

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.     

Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase

Methylglyoxal (MGO) is a highly reactive alpha-oxoaldehyde formed endogenously in numerous enzymatic and nonenzymatic reactions. The reactions between MGO and various amino residues in proteins not only result in inactivation of enzymes, but also lead to the formation of different detrimental advanced glycation endproducts (AGEs). Recently, it was reported that creatine kinase (CK, EC 2.7.3.2) activity could be reduced or even lost under incubation with MGO in vitro. In this study, an efficient CE analytical method was developed for the evaluation of CK activity. Based on this CE method, the inhibitory effect of MGO on CK activity was confirmed. Several MGO scavengers such as aminoguanidine (AG) and some thiols showed obvious protective effects on CK activity against MGO. Furthermore, tiopronin (TP), a hepatoprotective drug, was found for the first time to counteract MGO-induced inhibition of CK activity in CK reaction. Meanwhile, TP also retained adenosine diphosphate (ADP) generation level in plasma treated with MGO, which implies that this drug may have potential protective effect on other enzymes which are associated with adenine nucleotide metabolism. Besides, the established CE approach can be utilized as a model for screening effective MGO scavengers by monitoring CK-catalyzed conversion between adenosine triphosphate and ADP.     

Continuous concentration of bacteria in a microfluidic flow cell using electrokinetic techniques

A novel method for the concentration of bacterial solutions is presented that implements electrokinetic techniques, zone electrophoresis (ZE) and isoelectric focusing (IEF), in a microfluidic device. The method requires low power (< 3e-5 W) and can be performed continuously on a flowing stream. The device consists of two palladium electrodes held in a flow cell constructed from layers of polymeric film held together by a pressure-sensitive adhesive. Both ZE and IEF are performed with carrier-free solutions in devices in which the electrodes are in intimate contact with the sample fluid. IEF experiments were performed using natural pH gradients; no carrier ampholyte solution was required. Experiments performed in buffer alone resulted in significant electroosmotic flow. Pretreatment of the sample chamber with bleach followed by a concentrated solution of cationic detergent effectively suppressed electroosmotic flow.     

Two new esterase D (ESD) variants revealed by isoelectric focusing in agarose gel

Using isoelectric focusing in thin-layer agarose gel (AGIF) with the narrow pH range of 4.5-5.4, a high resolution of esterase D (ESD) isozyme banding patterns has been achieved. Some variant phenotypes which could not be distinguished from common ESD types by conventional electrophoresis have shown different patterns after AGIF. The IEF method permitted the distinction of two further variants in the ESD system, tentatively named ESD Rehren and ESD Ravensburg. We recommend, therefore, that for the classification of ESD phenotypes a high resolution IEF technique should be used.     

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.     

Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non‐denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non‐denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF‐separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme‐ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme‐ovotransferrin complex, can be effectively separated and extracted using non‐denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.     

A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell

We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis. These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions. The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection. The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots. The Protean IEF cell had the next highest number of spots detected per gel. In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel. Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed. A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed.     

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

Use of rotofor preparative isoelectrofocusing cell in protein purification procedure

The Rotofor cell is a preparative isoelectric focusing (IEF) apparatus, in which IEF is performed entirely in free solution. Electrofocusing in Rotofor cell has been described as well-suited for use at any stage of a purification scheme. However, it has some important limitations in resolving complex mixtures of proteins. This paper describes the advantages and disadvantages of using the Rotofor cell in purification protocols.