A nanoelectronics-blood-based diagnostic biomarker for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

There is not currently a well-established, if any, biological test to diagnose myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The molecular aberrations observed in numerous studies of ME/CFS blood cells offer the opportunity to develop a diagnostic assay from blood samples. Here we developed a nanoelectronics assay designed as an ultrasensitive assay capable of directly measuring biomolecular interactions in real time, at low cost, and in a multiplex format. To pursue the goal of developing a reliable biomarker for ME/CFS and to demonstrate the utility of our platform for point-of-care diagnostics, we validated the array by testing patients with moderate to severe ME/CFS patients and healthy controls. The ME/CFS samples' response to the hyperosmotic stressor observed as a unique characteristic of the impedance pattern and dramatically different from the response observed among the control samples. We believe the observed robust impedance modulation difference of the samples in response to hyperosmotic stress can potentially provide us with a unique indicator of ME/CFS. Moreover, using supervised machine learning algorithms, we developed a classifier for ME/CFS patients capable of identifying new patients, required for a robust diagnostic tool.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Interaction of hexokinase with the outer mitochondrial membrane and a hydrophobic matrix.

The major portion of rat brain hexokinase (HK type 1) is bound to the outer membrane of mitochondria and glucose-6-phosphate (G6P) can release the bound enzyme. In an attempt to look at the 'hydrophobic' component of binding, interaction of the enzyme with a purely hydrophobic matrix, palmityl-substituted Sepharose-4B (Sepharose-lipid) was investigated. Hexokinase readily bound to this matrix with retention of its catalytic activity. Glucose-6-phosphate which has a releasing effect on the mitochondrially bound enzyme, enhanced binding of the enzyme on the hydrophobic matrix. Chymotrypsin treatment of hexokinase which causes loss of binding to mitochondria, also results in loss of adsorption to the hydrophobic matrix, thus demonstrating that the 'hydrophobic tail' present at its N-terminal end is essential for binding in both cases. Data presented provide some new information relevant to understanding how hexokinase interacts with its natural binding matrix, the mitochondrion.

Use of reversible denaturation for adsorptive immobilization of urease.

Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization and enzymatic activity with lowering of Km (apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore, the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports.

Chemical modification of bacterial alpha-amylases: changes in tertiary structures and the effect of additional calcium.

A comparative study was performed on the effect of calcium on native and chemically modified forms of mesophilic and thermophilic alpha-amylases. Circular dichroism (CD) and irreversible thermoinactivation studies were carried out in the absence and presence of 10 mM calcium. From the CD experiments, changes in the tertiary structure of these enzymes, brought about by modification, were concluded. Furthermore, these changes were found to be influenced by the presence of calcium. Sorbitol was very effective in affording protection against irreversible thermoinactivation of native and modified forms of the enzymes, both in the absence and presence of calcium. Results are discussed in terms of the usefulness of this new approach involving a combination of medium and chemical modification for protein stabilization and enhancement of catalytic potential.

Proteolysis of mesophilic and thermophilic alpha-amylases: a comparative study.

A comparative study was performed on limited and extensive proteolysis of mesophilic (from Bacillus amyloliquefaciens [BAA]) and thermophilic (from Bacillus licheniformis [BLA]) alpha-amylases using trypsin. As expected, the thermophilic enzyme showed greater resistance to digestion by the protease. While the catalytic potential of BLA was enhanced on proteolysis, that of BAA was diminished owing to this process. Combined with greater catalytic activity, a lower thermal stability was observed for BLA on proteolytic treatment. For both enzymes, the extent of proteolytic cleavage was reduced in the presence of various stabilizing agents. The digestion patterns are explained in terms of available information in the literature on the structure of these proteins, especially in relation to segmental mobility.

Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability.

Chemical modification of lysine residues in two bacterial alpha-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 +/- 1 residues in BAA and 10 +/- 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.

Comparative studies on a mesophilic and a thermophilic alpha-amylase.

A comparative study was performed on thermal stability of mesophilic and thermophilic alpha-amylases, and the effects of various denaturing agents, organic solvents, and stabilizers were investigated. As expected, the thermophilic enzyme showed higher resistance toward denaturation in water as its natural medium, but such a difference could not be detected in nonaqueous environments. Furthermore, stability of these molecules was improved by including various stabilizing agents. Of the compounds tested, sorbitol provided the highest degree of protection, which was found to be owing to its effect on increasing Tm and its ability in totally preventing deamidation of amino acid residues in the protein molecules.

Nickel-induced substrate inhibition of bovine liver glutamate dehydrogenase.

The effects of nickel ions on reductive amination and oxidative deamination activities of bovine liver glutamate dehydrogenase (GDH) were examined kinetically by UV spectroscopy, at 27 degrees C, using 50 mM Tris, pH 7.8, containing 0.1 M NaCl. Kinetic analysis of the data obtained by varying NADH concentration indicated strong inhibition, presumably due to binding of the coenzyme to the regulatory site. In contrast, almost no inhibition was observed in the forward reaction. The fact that nickel ions have the capacity to enhance binding of NADH to the enzyme was confirmed by an electrochemical method using a modified glassy carbon electrode. Use of NADPH instead of NADH showed only a weak substrate inhibition, presumably related to lower affinity of NADPH for binding to the regulatory site. Lineweaver-Burk plots with respect to alpha-ketoglutarate and ammonium ions indicated substrate and competitive inhibition patterns in the presence of nickel ions, respectively. ADP at 0.2 mM concentration protected inhibition caused by nickel. These observations are explained in terms of formation of a nickel-NADH complex with a higher affinity for binding to the regulatory site in GDH, as compared with the situation where nickel is not present. Such effects may be important for regulation of GDH and other NADH-utilizing enzymes.

Adsorptive immobilization of erythrocyte membrane.

Immobilization of human erythrocyte membrane was carried out by adsorption on Fractosil, a porous form of silica. Acetylcholinesterase (AChE) was chosen as a representative membrane enzyme in this study. Dependency of adsorption on membrane concentration was determined. Positive cooperative interactions that occurred in the process of immobilization increased stability. Presence of hydrophobic ligands on derivatized Fractosil was found to enhance stability of immobilized preparations making them more effective for use in continuous catalytic transformations. It is suggested that adsorptive immobilization of membrane structures such as the human erythrocyte membrane fragments on Fractosil and other inexpensive supports may provide a convenient procedure for utilization of their catalytic potential. Such preparations may be used in diagnostic kits or for construction of biosensors.

Hexokinase 'binding sites' of normal and tumoral human brain mitochondria.

Interaction of type I hexokinase (HK-I) with the mitochondria obtained from the biopsy specimens of normal and tumoral human brain tissues was studied in the present investigation. This effort was undertaken with the aim of exploring possible differences in the mode of association of the enzyme with the outer mitochondrial membrane in the described sources. Results indicate that the two 'sites' for binding of HK-I suggested in the literature, based on extensive studies carried out on rat brain mitochondria, are similarly present in the human brain mitochondria. Differences in the microenvironments of HK binding, as reflected by the presented data, are suggested to be of importance in regulation of the catalytic potential of the bound enzyme. The real metabolic significance of this association in relation to cancer and its practical importance would need further investigation.

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization.

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.

Adsorptive immobilization of submitochondrial particles on Fractosil.

(1) Submitochondrial particles prepared from beef liver mitochondria were immobilized on Fractosil, a porous form of silica, in order to stabilize their enzymatic activity. (2) The catalytic activity of succinate-cytochrome c reductase, an enzyme complex of the inner mitochondrial membrane, was followed in this study. Adsorption resulted in significant stabilization with a lowering of K(m) (app.) for succinate, in spite of mass transfer and diffusion limitations expected to occur in such a complex and heterogeneous system. An increase in catalytic potential was also observed upon immobilization. These observations, taken together, suggest that substantial degree of conversation of substrates to their respective products may be achieved by such immobilized preparations. (3) Positive cooperative interactions for binding of submitochondrial particles to the matrix was observed, apparently with two sets of sites, the second set indicating a much greater hill coefficient. (4) The present report indicates that adsorption with the use of a porous inorganic support such as Fractosil may provide a simple and efficient method of immobilization. Such preparations containing membrane enzymes in their native microenvironments would be useful for continuous catalytic transformations and also for construction of biosensors.

Adsorptive immobilization of submitochondrial particles on concanavalin A Sepharose-4B.

Submitochondrial particles (SMPs) prepared from beef liver mitochondria were immobilized on concanavalin A Sepharose-4B (Con A-Sepharose). The process of immobilization was optimized by choosing an appropriate buffer system containing Mn2+ and Ca2+. Adsorption of SMPs on Con A-Sepharose was found to be reversible process, nonelectrostatic in nature, and responsive to the presence of methyl alpha-d-glucopyranose and alpha-d-mannose. The involvement of membrane glycoproteins in the adsorption process was thus demonstrated. Further analysis of the data obtained on competition of binding by sugar molecules provided competition constants reflecting the potency of inhibition by each individual carbohydrate. Positive-cooperative interactions for binding to the matrix were observed especially at high concentrations of SMPs. The immobilized preparations were used successfully in continuous catalytic transformations involving succinate-cytochrome c reductase (SCR) and enzyme complex of the inner-mitochondrial membrane. Best results were obtained when such operations were carried out at 37 degrees C.

Reversible association of ox liver glutamate dehydrogenase with the inner mitochondrial membrane.

A comparative study on the catalytic and allosteric properties of particulate and soluble forms of ox liver glutamate dehydrogenase has been carried out. The response of the bound enzyme to release by various effectors was investigated. The particulate enzyme was found to have catalytic activities similar to the free enzyme in contrast to its behaviour when bound to pure anionic phospholipids. Possible reasons for such outstanding differences are discussed.

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme.

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).

Use of hexadecyl Fractosil as a hydrophobic carrier for adsorptive immobilization of proteins.

Fractosil, a porous form of silica, has been used for the preparation of a hydrophobically derivatized carrier for protein immobilization. Interaction of a number of arbitrarily chosen proteins with hexadecyl-substituted Fractosil has been investigated. Binding of proteins was found to take place with retention of their native properties. Glutamate dehydrogenase, used as a model allosteric protein, was found to retain its catalytic and allosteric properties upon binding to the adsorbent in the form of suspension or column. Positive cooperative interactions for binding of bovine serum albumin and glutamate dehydrogenase to the matrix were observed. These findings are discussed in terms of hydrophobic interactions occurring between various residues of the protein molecules and the hydrophobic ligands in addition to those interactions which may occur with the unsubstituted gel. Results presented on immobilized glutamate dehydrogenase, trypsin, alpha-chymotrypsin, alpha-amylase, and amyloglucosidase clearly indicate possible potential of the support for continuous catalytic transformations.

Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site.

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) is inhibited by acidic phospholipids such as phosphatidylinositol, phosphatidylserine, and cardiolipin. Several aspects of this inhibition are atypical when compared to inhibition by established reversible inhibitors of this enzyme such as the product, Glc-6-P. Maximal inhibition is attained rather slowly (approximately 30 min at 22 degrees C), and is not reversed by simple dilution of the enzyme-lipid mixture. Ligands such as ATP or Glc-6-P can protect the enzyme against inhibition by acidic phospholipids; addition of protective ligands after mixing of enzyme and lipids does not, however, reverse inhibition that occurred prior to ligand addition. Inhibition can be prevented but not reversed by elevated (0.1-0.2 M) [NaCl], indicating a probable role for electrostatic forces in the interaction of lipid with enzyme. Greater inhibition is seen at 22 degrees C than at 3-4 degrees C, suggesting that hydrophobic interactions may also be involved. It is suggested that acidic phospholipids inhibit brain hexokinase by binding at the nucleotide-binding site of the enzyme. The effectiveness of ATP (or the ATP analog, Cibacron Blue) in protecting against inhibition by acidic phospholipids is attributed to direct competition between ATP and the phospholipid for a common binding site. The effectiveness of Glc-6-P (or analogs) in preventing the inhibition is attributed to a conformational change, induced by the binding of this ligand, which prevents binding of ATP or acidic phospholipids to the enzyme. The pH dependency of the inhibition has suggested involvement of the protonated form of a dissociable group (pK approximately 7) on the enzyme in the interaction with acidic phospholipids; this may be the histidyl residue implicated by Solheim and Fromm [Biochemistry 19, 6074-6080 (1984)] in the binding of ATP to brain hexokinase. Structural similarities in the nucleotide-binding sites of several nucleotide-binding enzymes suggest that similar inhibition by acidic phospholipids may be seen with other enzymes of this type; there are already some reports to this effect.

Interaction of proteins with Triton X-100-substituted sepharose 4B.

Interaction of a number of arbitrarily chosen proteins with Triton X-100-substituted Sepharose 4B has been investigated. Of the proteins examined, bovine serum albumin, hemoglobin, glutamate dehydrogenase, and pepsin were found immobilized on the adsorbent. Binding of these proteins occurred irrespective of pH and NaCl concentration. Cytochrome c, used as a model protein, was totally immobilized only at low pH. Adsorption of glutamate dehydrogenase and pepsin took place with retention of their catalytic activities. Moreover, glutamate dehydrogenase used as a model allosteric enzyme, was found to retain its native properties upon binding to the adsorbent in the forms of suspension or column. Results are discussed in terms of specific interactions involving the hydrophobic region of Triton X-100 and the apolar patches or crevices present on the surface of protein molecules. Possible potential of the matrix as a method for preparation of biologically active immobilized proteins and its application in continuous operations are also discussed.

Deficient lipid-protein interaction in mitochondrial cytochrome oxidase from newborn and old rat brain.

In contrast to the mature brain, Arrhenius plots of mitochondrial cytochrome c oxidase from newborn and old rat brain show no breaks (transition temperature); the apparent activation energy of the enzyme, in the physiological range of temperature, is also significantly higher. The results indicate deficient lipid-protein interaction as well as differential fluidity status in the inner mitochondrial membranes of the newborn and aged brain. These changes may contribute to the known deficiency in oxygen consumption of the newborn and old brain.