Preparative Flat-bed Isoelectric Focusing Research Articles

Convergence of amino acid compositions of certain groups of protein aids in their identification on two-dimensional electrophoresis gels.

The amino acid composition (AAC) versus the protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat-bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root-mean-square rule (Rmsd) with measures the difference between the AAC of chosen protein versus those in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained within 1.5-1.0 with m +/- 3kDA and pI +/- 4. Certain groups of multidomain proteins have quasi-unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC-PI method may become an additional search tool for protein functions.

Evidence for the lack of exo-cellobiohydrolase activity in the cellulase system of Trichoderma reesei QM 9414

A cellulolytic enzyme (pI 4.3) was isolated from culture filtrates of Trichoderma reesei QM 9414 by column chromatography and preparative flat bed isoelectric focusing. The focusing experiments were carried out using an extremely shallow pH gradient (pH 3.7–4.6), which made it possible to recover one single protein band out of numerous Trichoderma isoenzymes and/or multi-enzyme complexes in the acidic pH region. The purified hydrolase is homogeneous as assessed by analytical isoelectric focusing, SDS gel electrophoresis, and titration curve analysis under detergent/ urea conditions. It was found to have an apparent molar mass of 68 000 g mol −1 (SDS-PAGE) and showed its optimum activity towards cellotetraose at pH 4 and 60°C. It is a glycoprotein with a carbohydrate content of 27% (as glucose). The hydrolase does not act with an endo-mechanism (CMC as substrate) and splits off mainly cellobiose from microcrystalline cellulose. In the current literature the enzyme would thus be termed an exo-cellobiohydrolase [1,4-β-D-glucan cellobiohydrolase (CBH), EC 3.2.1.91]. However, when the enzyme's action pattern was further evaluated using defined soluble cellodextrins (DP 2–7) as substrates, it became obvious from the recorded concentration-time course data that the enzyme does not exclusively split off cellobiose units from the oligomers. In conclusion, we do not believe that a cellobiohydrolase analogous to β-amylase (EC 3.2.1.2) exists, at least in the cellulase system of Trichoderma reesei. The term CBH or cellobiohydrolase should thus not be used any longer in the literature.

Isolation from lugworm (arenicola cristata) of four proteases that activate cyclic amp phosphodiesterase

1. 1. Four cyclic AMP phosphodiesterase-activating activities, designated as A, B, C and D, were isolated from lugworm, Arenicola cristata, by preparative flat-bed isoelectric focusing. Activators C and D were further purified by TSK 3000SW HPLC to homogeneity. 2. 2. Activators A, B, C, and D corresponded to pls of 4.4, 5.0, 5.2 and 5.4; their mol wts were estimated to be 36,200, 30,500, 30,200 and 28,300 respectively. 3. 3. The protease nature of these activities were confirmed by the inhibition by several trypsin inhibitors of their activation of phosphodiesterase and by their hydrolysis of TAME, a synthetic trypsin substrate. Only protease A also hydrolyzed BTEE, a chymotrypsin substrate.

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.

Purification of human myoglobin by preparative flat bed isoelectric focusing

AbstractWe have prepared a standard of human myoglobin by flat bed isoelectric focusing, to be used in immunological and chemical methods of measurement in clinical biology. Myoglobin was extracted from human psoas or myocardium and then purified by preparative isoelectric focusing in a granulated gel. The purified myoglobin was examined by analytical isoelectric focusing, high performance liquid chromatography (HPLC), immunoelectrophoresis, and two‐dimensional gel electrophoresis. Myoglobin was quantified by three methods: (i) Drabkin's method, and by the determination of iron using (ii) the bathophenanthroline method, and (iii) the atomic emission method. There was good correlation between all three methods. Purified myoglobin was stabilized by conversion to the cyanmetmyglobin form, and stored until use at 4 °C (± 2 °C). or at – 20 °C in the liquid state after addition of ethylene glycol.

Chromatofocusing of skin fibroblast sphingomyelinase: Alterations in Niemann-Pick disease type C shared by G M1-gangliosidosis

Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by Chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with G M1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.

Immunological properties of rat lens gamma-crystallins. I. Characterization of the major components.

Rat lens gamma-crystallins were separated by preparative flat bed isoelectric focusing (pH 7-11). The fractions obtained were submitted to analytical isoelectric focusing, fused rocket immunoelectrophoresis and immunodiffusion. The results demonstrate that rat lens gamma-crystallins are electrophoretically and immunologically heterogenous. Evidence is given that there are two immunologically different groups. Rat gamma 1-crystallin, the prominent and immunologically homogeneous part, is identical to calf lens gamma-crystallin. The second group of proteins, gamma 2-crystallins, is heterogeneous and shows only partial identity with gamma 1-crystallin.

Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by molecular sizing chromatography and preparative flatbed isoelectric focusing.

The soluble portion of a large particle fraction which was derived from the muscle larva of T. spiralis was subjected to molecular sizing column chromatography using Sephacryl S-200. Five major peaks of 280 nm absorbing material were obtained. Analysis by immunoelectrophoresis revealed that each peak contained antigens, with the majority of them occurring in peaks 3, 4 and 5. Preliminary studies indicated that peak 4(mol. wt range 20 000--10 000) contained protection-inducing antigens. Crossed-immunoelectrophoretic and single-dimension electrophoretic analysis of peak 4 revealed a minimum of 10 antigens, while analytical isoelectric focusing demonstrated the presence of proteins with widely different pl, ranging from 4.0 to 9.0. Peak 4 was fractionated by preparative flatbed isoelectric focusing (PIEF) using two gradients: one from 3.5 to 9.5 and the other from 3.5 to 5.5. Fused rocket immunoelectrophoretic (FRIEP) analysis of both runs indicated that several antigens were separated from the others: one at pl 4.0 and the other at pl 9.0. The remaining antigens focused between pl 4.3 and 4.9. One hundred micrograms of whole peak 4, pl 9.0 antigen and the group of antigens at pl 4.3--4.9 were each separately injected, along with Freund's complete adjuvant, into mice. In addition, a portion of the pl 4.0 antigen was also assayed for protection. All antigenic preparations induced significant levels of protection. The pl 4.0 was further analysed on high-performance liquid chromatography (HPLC). Two sharp peaks of antigen, as detected by FRIEP, were eluted isocratically with 65% acetonitrile from a C-18 (aliphatic) column. Both peaks of antigen showed complete cross-reactivity on FRIEP and absorbed at 220 nm. Amino acid analysis of each HPLC peak revealed no detectable differences in composition. Each peak contained predominance of aspartic (13 mol%) and glutamic (18 mol%) acid. This antigen did not contain significant quantities of aromatic amino acids, and absorbed strongly at 206 nm. Neither the pl 4.0 or pl 9.0 antigen stained positively with the PAS reaction.

Agarose‐Sephadex: A new improved matrix for preparative flatbed isoelectric focusing

AbstractA new, simple procedure is described for the preparation gel matrix for preparativde isolelectic focusing (IEF). 0.5–1% Agarose of IEF grade is incorporated with 2.5% Sephadex G‐200 Superfine (SF) and 3% Pharmalyte, yielding a uniform and rifid m atirx. No gel washing, swelling of degassing is necessary. Optimum gel consistency is quickly and easily obtained since no dehydration of the gel timum gel consistency is quickly and easily obtained since no dehydration of the gel slurry is required. Gel preparation time is reduced to 0.5–1 h and gel handling is simplified. Protein can be easily recovered from the matrix. The pH gradient profile and resolving properties of agarose‐Sephadex were compared with those of Spphdex G200 SF and IEF‐grade agarose. The profiles of agarose‐Sephadex and Sephadex matrixes are smoother and extend further in both directions than those for agarose. Focusing in agarose‐Sephadex and Sephadex also results in fewer edge effects. The ability to distinguish closely resolved bands in agarose‐Sephadex is superior due to its ability to be directly stained and its avoidance of eletroen‐domestic effects associated with focusing in sgarose. Changes in voltages enables us to follow the development, stability, and decay of the pH gradient. Agarose‐Sephadex and Sephadex pH gradients developed more smoothly and remained more stable than those for agarose. This new support system combines the good focusing properties of Sephades G‐200 SF with the simple handling, rapid and uniform gel formation of agarose, while overcoming many of the drawbacks of using either material alone.

Immunological characterization of calf lens γ-crystallins, separated by preparative isoelectric focusing

Calf gamma-crystallins were separated by preparative flat-bed isoelectric focusing and characterized according to their immunological properties, molecular weights and isoelectric points. Immunologically 4 different groups of gamma-crystallins (gamma 1a-1d-crystallin) could be detected. The main immunological component, the gamma 1b-crystallin corresponds to two polypeptides with molecular weights of 20,500 and 22,000. The two minor components, the gamma 1c- and gamma 1d-crystallin, are immunologically partial identical with the main component gamma 1b-crystallin. One of these minor components, the gamma 1d-crystallin, has a MW of 29,000. The 4th antigen (gamma 1a-crystallin), which has a MW of 21,000, seems to have only remote relationship with the major gamma-crystallin component.

Hydrolysis of some cystine amniopeptidase and aminopeptidase substrates by human placental enzymes after isoelectric focusing.

Several cystine aminopeptidase- and aminopeptidaselike activities of mature human placenta were demonstrated by chromatography on CM-Sephadex and subsequent preparative flat bed isoelectric focusing. Enzyme tests were carried out by using a micro modification of the Bratton-Marshall reaction with seven amino acid-rho-nitroanilides and one amino acid beta-naphthylamide as substrates. By this method three main activities were detected; each was nonhomogeneous and capable of hydrolyzing cystine aminopeptidase- as well as aminopeptidase substrates. The substrates benzyl-cystine-rho-nitroanilide, cystine di-rho-nitro-anilide and cystine di-beta-naphthylamide were split by different enzymes. By studying the effects of several effectors or of heating, no enzyme with the properties described in the literature for serum cystine aminopeptidase could be unequivocally demonstrated. The results report here suggest that serum cystine aminopeptidase--in contrast to placenta cystine aminopeptidase--is either altered severely in its structure or that it represents a multiplicity of enzymes, which attack "specific" substrates and are therefore jointly manifested as cystine aminopeptidase.

Separation and determination of the relative concentrations of the homogeneous components of soybean leghemoglobin by isoelectric focusing

The multiple components of soybean ferric leghemoglobin are readily separated by analytical and preparative flat bed isoelectric focusing in both the presence and also the absence of the ligand nicotinate. In the presence of nicotinate the separation by isoelectric focusing is more rapid and results in sharper bands of the very stable ferric leghemoglobin nicotinate complexes. The separation is sensitive enough to permit analytical experiments on leghemoglobin from single nodules. Leghemoglobins a and c 1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c 2 prepared by ion exchange chromatography is an approximately 1 : 2 mixture of leghemoglobins c 2 and c 3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c 3 content increases dramatically as very young nodules mature. The increase in relative leghemoglobin a content suggests that leghemoglobin a might be required for regulation of nodule O 2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c 1 content to leghemoglobin c 3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c 2 content to leghemoglobin c 3 content increases slowly throughout nodule development. Ratios of leghemoglobin b content to leghemoglobin a content and of total leghemoglobin d content to total leghemoglobin c content were almost independent of nodule age. Leghemoglobins a and b might be related biosynthetically, as might leghemoglobins c and d.