Refinement of noncalorimetric determination of the change in heat capacity, DeltaC(p), of protein unfolding and validation across a wide temperature range

The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.     

The denaturing action of lysophosphatidylcholine as studied by calorimetric and rheological techniques

The potency of lysophosphatidylcholine to perturb protein structure was investigated by differential scanning calorimetry and rheological measurements using myosin as a model protein. At physiological ionic strength (0.15 M NaCl) 5mM lysophosphatidylcholine produced a detectable reduction in the protein's enthalpy of denaturation, while concentrations less than or equal to 2 mM had no effect. At higher salt concentrations (0.6 M NaCl) lower concentrations of lysophosphatidylcholine were needed in order to reduce the enthalpy of denaturation. Also, the changes in myosin conformation, as judged from calorimetric measurements, became more extensive as the incubation temperature for myosin-lysophosphatidylcholine systems was increased from 10 degrees to 30 degrees C. Rheological techniques allowed detection of changes in the structure of filaments of myosin (in 0.15 M) upon addition of 0.2 mM lysophosphatidylcholine. The denaturing action of lysophosphatidylcholine is compared to the more familiar detergent sodium dodecyl sulphate.     

Limited Tryptic Degradation of Urea Denatured Glycinin

Digestibilities of native, 5 m urea-denatured and 8 m urea-denatured glycinin were studied. Urea was removed by dialysis before digestion. The tryptic digestion of the proteins are influenced by ionic strength. Under low ionic strength condition (0 m NaCl), the proteins, even native glycinin, are well degraded. On the other hand, under high ionic strength condition (0.5 m NaCl), native glycinin resists the tryptic attack and 5 m urea-denatured glycinin is best degraded. The digestibility of 8 m urea-denatured glycinin is lower than that of 5 m urea-denatured one under the condition. The gel filtration and electrophoretic properties show that the digestion intermediate like glycinin-T (the intermediate from native glycinin) is contained in the digestion products. These suggest that the urea-denatured protein contains the almost renatured component after removal of urea. A larger amount of the glycinin-T-like protein was detected at 8 m urea denaturation than at 5 m urea. Therefore, glycinin renatures more readily from 8 m urea denaturation. Probably this is the cause of the decreased digestibility at 8 m urea denaturation.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Microcalorimetric study of heat denaturation of DNA from calf thymus DNA in the absence of magnesium ions

Thermal denaturation was studied for a wide range of magnesium ions concentrations and salt concentration 0.15 M NaCl. It was shown that thermal stability of DNA increases at low Mg/2P ratios and decreases at high concentrations of magnesium ions. Up to Mg/2P = 10 DNA denaturation is an equilibrium process. With an increase in magnesium ions concentrations the enthalpy of DNA denaturation reaches the maximum at Mg/2P = 10 (50 kJ/mole base pairs). DNA aggregation and appearance of a new heat absorption peak is observed in the high temperature region at Mg/2P = 10. At this region of magnesium ions concentrations DNA denaturation process is non-equilibrium.     

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.     

Thermodynamic stability of porcine beta-lactoglobulin. A structural relevance.

The proposed biological function of beta-lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine beta-lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high-sensitivity differential scanning calorimetry within the pH range 2.0-10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 +/- 0.6 kJ.mol-1. K-1,which is close to the denaturation heat capacity increment DeltadCp = 9.6 +/- 0.5 kJ.mol-1.K-1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 degrees C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ.mol-1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two-state model of denaturation. Based on the pH-dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, DeltadGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH-dependence of DeltadGE is analysed in terms of the contributions of side-chain H-bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.     

Stability of recombinant Lys25-ribonuclease T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Some thermodynamic implications for the thermostability of proteins

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* approximately 283K, independent of the heat denaturation temperature, T(m). This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of approximately 0.008 kJ/mole/residue is, on average, associated with a 1 degrees C increase in T(m). An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.     

Carnosine promotes the heat denaturation of glycated protein

Glycation alters protein structure and decreases biological activity. Glycated proteins, which accumulate in affected tissue, are reliable markers of disease. Carnosine, which prevents glycation, may also play a role in the disposal of glycated protein. Carnosinylation tags glycated proteins for cell removal. Since thermostability determines cell turnover of proteins, the present study examined carnosine's effect on thermal denaturation of glycated protein using cytosolic aspartate aminotransferase (cAAT). Glycated cAAT (500 microM glyceraldehyde for 72h at 37 degrees C) increased the T(0.5) (temperature at which 50% denaturation occurs) and the Gibbs free energy barrier (DeltaG) for denaturation. The enthalpy of denaturation (DeltaH) for glycated cAAT was also higher than that for unmodified cAAT, suggesting that glycation changes the water accessible surface. Carnosine enhanced the thermal unfolding of glycated cAAT as evidenced by a decreased T(0.5) and a lowered Gibbs free energy barrier. Additionally, carnosine decreased the enthalpy of denaturation, suggesting that carnosine may promote hydration during heat denaturation of glycated protein.     

Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase

Chemical modification of carboxyl groups of glucoamylase from a mesophilic fungus, Fusarium solani, was carried out using ethylenediamine as nucleophile in the presence of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Modification brought about a dramatic enhancement of catalytic activity and thermal stability of glucoamylase. Temperature and pH optima of ethylenediamine-coupled glucoamylase (ECG) increased as compared with those of native enzyme. The specificity constant (k(cat)/K(m)) of native, ECG-2, ECG-11, and ECG-17 was 136, 173, 225, and 170, respectively, at 55 degrees C. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms. All of the modified forms were stable at higher temperatures and possessed high Delta G* against thermal unfolding. The effects of alpha-chymotrypsin and subtilisin on the modified forms were activating as compared with native. Moreover, denaturation of ECG-2, ECG-11, and ECG-17 in urea at 4 mol x L(-1) also showed an activation trend. A possible explanation for the thermal denaturation of native and increased thermal stability of ECG-2, ECG-11, and ECG-17 at higher temperatures is also discussed.     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.