Electrophoretic methods for studying protein-protein interactions.

Protein-protein interactions are involved in many biological processes ranging from DNA replication, to signal transduction, to metabolism control, to viral assembly. The understanding of those interactions would allow the effective design of new drugs and further manipulation of those interactions. Several useful analytical methods are available for the study of protein-protein binding, and among them, electrophoresis is commonly used. We describe two types of electrophoresis: gel electrophoresis and capillary electrophoresis. Gel electrophoresis is a well-established method used to study protein-protein interactions and includes overlay gel electrophoresis, charge shift method, band shift assay, countermigration electrophoresis, affinophoresis, affinity electrophoresis, rocket immunoelectrophoresis, and crossed immunoelectrophoresis. These techniques are briefly described along with their advantages and limitations. Capillary electrophoresis, on the other hand, is a relatively new method and affinity capillary electrophoresis has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of stoichiometry of biomolecular interactions. It offers short analysis time, requires minute amounts of protein samples, usually involves no radiolabeled compounds, and, most importantly, is carried out in solution. We summarize the principles of affinity capillary electrophoresis for studying protein-protein interactions along with current limitations and describe in depth its application to the determination of stoichiometries of tight and weak binding protein-protein interactions. The protocol presented in the experimental section details the use of affinity capillary electrophoresis for the determination of stoichiometry of protein complexes.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

Cell electrophoresis research directed toward clinical cytodiagnosis.

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.     

Profile--Richard A. Mathies. Interview by Suzanne Berry.

Richard A. Mathies (Fig. 1) is a professor of chemistry at the University of California (UC) at Berkeley. His early work at UC was on the use of resonance Raman and time-resolved optical spectroscopy to elucidate the structure and reaction dynamics of energy and information-transducing photoactive proteins called rhodopsins. His work on the Human Genome Project led to the development of high-throughput platform technologies including capillary array electrophoresis and energy transfer fluorescent dye labels for DNA sequencing and analysis. He has also pioneered the development of microfabricated capillary electrophoresis devices, capillary array electrophoresis microplates and microfabriated integrated sample preparation and detection methods. He is the co-founder of the Center for Analytical Biotechnology at UC Berkeley. Mathies was interviewed at the BIOMEMS and Biomedical Nanotechnology conference in Columbus, Ohio, 21-25 September 2001, where he gave a talk about capillary array electrophoresis-based microprocessors. Such devices could be used as point-of-care clinical and genetic analyzers, in integrated microfluidic sequencing chips and in DNA-based computing.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

A peptide mapping technique--a three map system.

A high resolution peptide mapping system has been described. The method utilizes three maps for separation of the acidic, basic, and neutral constituents of a proteolytic digest of a protein. Peptides from a proteolytic digest are separated by electrophoresis at pH 6.5 into acidic, basic, and neutral peptides. The secondddimensional electrophoresis is performed at pH 2.1 for each of acidic peptides and basic peptides. The third map is prepared from the neutral peptides. For this map, paper chromatography was used for the first dimension and electrophoresis at pH 2.1 for the second dimension.     

Acid-base and optical properties of heparin.

Sodium dodecylsulfate (SDS) can be removed from protein by gel electrophoresis. This principle is useful for separating protein bands which are close to each other in SDS gel electrophoresis. We accomplished this by “two-stage” gel electrophoresis. In this system, SDS gel electrophoresis was carried out as the first step. Gel electrophoresis was then continued (after replacing the buffer) without SDS. SDS was then eluted from the gel into the lower buffer during the second stage. Separation of the subunits was significantly improved relative to simple SDS gel electrophoresis.     

Steroid receptors in the human prostate. 1. Estradiol - 17beta binding in benign prostatic hypertrophy.

Agar gel electrophoresis and ultracentrifugation on continuous sucrose gradients revealed the presence of a 4S estradiol 'receptor' in cytosols of samples of human benign hyperplastic prostate tissue. High affinity (charcoal stability) and saturability (disappearance with excess competitor) binding characteristics of the molecular species concerned facilitated its clear distinction from similarly migrating serum albumin-steroid complexes. Our data, obtained with human serum, purified human albumin and albumin-enriched cytosol strongly suggest that agar gel electrophoresis, when used alone, may lack specificity for the quantification of estrogen 'receptors'. Radioligand binding to these molecules may be obscured by similarly migrating albumin-steroid complexes which fail to dissociate during electrophoresis. We advocate the inclusion of competitor experiments to improve the specificity of the method.     

Monitoring solution-phase combinatorial library synthesis by capillary electrophoresis.

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations.     

Minimal electrophoresis time for DNA sequencing.

This study presents a mathematical approach that allows one to determine the shortest electrophoresis time and migration path length required for DNA sequencing. The calculation was applied to the capillary electrophoresis of a DNA sequencing separation and showed that acceptable resolution could be obtained using a shorter path length than anticipated.     

Separation of allelic variants by two-dimensional electrophoresis.

The resolving power of two-dimensional (2-D) gel electrophoresis has been tested using 17 allele products at five loci. Standard O'Farrell gels could separate 13 of these isozymes. The addition of a second pH gradient made it possible to separate all but one of the variant proteins. These results indicate that 2-D gel electrophoresis can resolve more than 90% of variants originally detected by one-dimensional (1-D) electrophoresis. The implications of these results for the low estimates of average heterozygosity obtained in surveys using 2-D gel electrophoresis are discussed.     

Analysis of the fibrin-polymerizing reaction using sodium dodecylsulfate-agarose gel electrophoresis.

1. A new method of sodium dodecylsulfate gel electrophoresis, sodium dodecyl-sulfate-agarose gel electrophoresis, was developed. The new electrophoresis was shown to have a high and efficient resolving power for fibrin polymers and was also applicable to other proteins. 2. By sodium dodecylsulfate-agarose gel electrophoresis, the fibrin polymerizing reaction with activated fibrin stabilizing factor was analyzed in detail. The cross-linking between the gamma-chains was indicated to occur at an intermolecular level. The solubility of polymerized fibrin was suggested to be related mainly to the formation of the crosslinks between gamma-chains.     

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.     

Semipreparative electrophoresis with prestained proteins.

A simple method for prestaining proteins with Coomassie blue G prior to acrylamidegel electrophoresis is described. Many, although not all, undenatured proteins can be stained. This prestaining technique can be conveniently coupled with a second electrophoresis step, using Sephadex as a supporting medium, to yield isolated protein in about 4 h.     

NADP-specific isocitrate dehydrogenase of Escherichia coli. IV. Purification by chromatography on Affi-Gel Blue.

Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) from Escherichia coli. The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature. The preparation was judged to be homogeneous by non-denaturing electrophoresis at pH 7.5 and denaturing electrophoresis in the presence of sodium dodecyl sulfate. The subunit molecular weight of 53 000, determined by sodium dodecyl sulfate gel electrophoresis, is in reasonable agreement with the value of 46 900 estimated from the amino acid composition data.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.     

Scrambling of bands in gel electrophoresis of DNA.

Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation of the gel. These data are in agreement with the results of a computer simulation based on a theoretical model of electrophoresis.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.