Change In Net Charge Research Articles

The electrophoretic mobility of DNA fragments differing by a single 3′‐terminal nucleotide in an automated capillary DNA sequencer

The electrophoretic mobility of DNA fragments that differ by a single 3'-terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5'-fluorescently labelled primer to generate DNA fragments with 3'-hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3'-terminal nucleotide. It was found that the 3'-terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) -C > -A > -T > -G (slower). The C-terminated fragments were the fastest and the G-terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed.

Influence of the Net Charge on the Reactivity of a Manganese(IV) Species: Leading to the Correlation of Its Physicochemical Properties with Reactivity

Clarifying how versatile physicochemical parameters of an active metal intermediate affect its reactivity would help to understand its roles in chemical and enzymatic oxidations. The influence of the net charge on electron transfer and hydrogen abstraction reactions of a manganese(IV) species having hydroxide ligand has been investigated here. It was found that increasing one unit of the positive net charge from 2+ to 3+ would accelerate its electron-transfer rate by 10–20 fold in oxygenation of tris(4-methoxyphenyl)phosphine. In contrast, the hydrogen abstraction rate is insensitive to its net charge change, and the insensitivity has been attributed to the compensation effect between the redox potential and pKa, which determine the hydrogen abstraction capability of a metal ion. Similar net-charge-promoted electron transfer but not hydrogen abstraction has also been observed in intramolecular electron transfer and hydrogen abstraction reactions when using thioxanthene as substrate. Together with the previous understanding of the reactivity of the identical manganese(IV) species having MnIV–OH or MnIV=O functional groups, the relationships of the oxidative reactivity of an active metal intermediate with its physicochemical parameters such as the net charge, the redox potential and the metal–oxygen bond order (M–O versus M═O) have been discussed with this manganese(IV) model.

Relationships Between Electrically Evoked Potentials and Loudness Growth in Bilateral Cochlear Implant Users

The goals of this study were (1) to describe the relationship between electrically evoked compound action potential (ECAP) and electrically evoked auditory brainstem response (EABR) amplitude growth functions and loudness growth functions in bilateral cochlear implant (CI) users, and (2) to determine whether matching the stimulus levels in the two ears of bilateral CI users based on equal ECAP amplitude, EABR amplitude, or current level resulted in the smallest discrepancy in loudness rating across the two ears. Ten adult, bilateral CI users participated in this study. The stimulus used to elicit loudness judgments and generate ECAP and EABR growth functions was a train of biphasic current pulses (32 μs/phase) presented at a rate of 23 pps. Loudness growth functions were obtained with a method of constant stimuli. ECAPs were measured using the implant telemetry system. EABR growth functions were recorded using surface electrodes and standard averaging techniques. Both ears of each subject were tested. For each ear, ECAP, EABR, and loudness functions were recorded using both an apical and basal stimulating electrode. Both the physiologic and psychophysical growth functions were fit using linear regression techniques. Comparison of the regression equations obtained for the two ears revealed that stimulus levels that yielded approximately equal ECAP amplitudes in the two ears were judged to differ in loudness, on average, by 20% for electrode 3 and 14% for electrode 13. Stimulation levels that evoked similar amplitude EABRs differed in loudness, on average, by 50% for electrode 3 and 13% for electrode 13. Matched stimulus current levels were judged to differ in loudness, on average, by 14% for electrode 3 and 16% for electrode 13. No significant differences in loudness discrepancy across ears derived from equal amplitude ECAP, EABR, or matched current levels were found. This study demonstrated that stimuli that evoke equal amplitude neural responses in both ears of a bilateral CI user or which are matched in current level cannot be assumed to be perceived as equally loud. No statistically significant differences in accuracy were found between ECAP, EABR, or matched current levels or between the basal and apical electrode in approximations of equal loudness.

D‐Loop Facilitates Hydride Transfer in Transhydrogenase

Transhydrogenase (TH) is located in the mitochondrial inner membrane and maintains NADPH levels to counteract oxidative stress resulting from oxidative respiration. Enzymes in the electron transport chain work to reduce oxygen to water and pump protons (H+) into the inter‐membranous space. The accumulation of H+ creates an electrochemical driving force, termed the proton motive force (pmf), which is used to power ATP synthase. However, the oxidative reactions also produce free radicals that are very damaging to cellular DNA and other molecules. To counteract this, the pmf powers TH to exchange hydride ions (H−) between two nicotinamide nucleotide cofactors, NAD(H) and NADP(H). TH is composed of three domains (DI, DII, and DIII). DII is membrane bound while DI and DIII are soluble, making them amenable to crystallization. DI and DIII are directly involved in hydride transfer between NAD(H) and NADP(H). The High Tech High SMART Team (Students Modeling A Research Topic) modeled TH (PDB: 1XLT, 1PNO) using 3D printing technology to visualize the hydride transfer. The models highlight the D‐loop in DIII in both open and closed conformations. The Dloop responds to changes in net charge when H− is transferred, causing DIII to move, which is linked to the inward transfer of H+ driven by the proton motive force. Hence, TH is able to make NADPH to counteract oxidative stress. Supported by a grant from the HHMI Pre‐College Program

Ultra Acidic Strong Cation Exchange Enabling the Efficient Enrichment of Basic Phosphopeptides

We present a straightforward method to enrich phosphopeptides with multiple basic residues, an under-represented class in common enrichment strategies. Our method is based on a two-dimensional strong cation exchange (SCX) strategy, operating at two different acidic pHs, enabling both separation and enrichment of different classes of phosphopeptides. The principle of enrichment is based on the change of net charge of phosphorylated peptides under strong acidic conditions in the second SCX, whereas the net charge of regular peptides remains unchanged, thus enabling separation based on net charge. Application of our tandem SCX approach to a modest amount of human cells allowed the identification of over 10,000 unique "basic" phosphopeptides of which many represent putative targets of basophilic kinases.

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers

The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

SU‐E‐J‐84: Radiation Induced Currents in RF Coil Conductors: Measurements and Simulations

Purpose: The integration of a medical linac with magnetic resonance imaging (MRI) has the potential to provide exquisite soft tissue contrast and real time imaging during the radiation treatment. However, during a real time treatment‐imaging session, the direct irradiation of MRIˈs radiofrequency (RF) coil by the pulsed x‐ray beam induces a current in the conductor of the RF coil due to the release of Compton electrons. This radiation induced current (RIC) can potentially degrade the SNR in acquired MRI images. The present work investigates methods of minimizing this RIC both experimentally and through Monte Carlo simulations.Methods: Copper and aluminum metal plates emulating the conductors used in RF coils were connected to an amplifier and placed in an RF cage. The plates (i.e. “detector”) were irradiated by the linacˈs pulsed 6 MV beam through the RF cage. The induced signal was measured by an oscilloscope and recorded using a PC. Various materials were used as buildup in an attempt to establish electronic equilibrium in the “detectors” ‐ thus removing the undesired RIC. A Monte Carlo script was written which counts the amount of charge entering and leaving a specified “detector volume”, and determines the net change in charge per primary history as a measure of RIC. The simulation geometry mimics the experimental setup.Results: It has been clearly demonstrated by both measurements and simulations that buildup of the same material as the conductor will reestablish electronic equilibrium and remove the RIC. Also, using a polymer with a density close to that of the conductor (i.e. Teflon with aluminum) for buildup will reduce the RIC to negligible amplitude Conclusions: With the proper combination of coil conductor and buildup, the RIC can be reduced to negligible amplitudes. Future work will assess the importance of RIC for the SNR in MRI images.

Thermochemical Treatment (Thermolysis) of Petrochemical Wastewater: COD Removal Mechanism and Floc Formation

Thermo-chemical precipitation (thermolysis) has been used as a pretreatment method for COD and color reduction from various industrial effluents. This paper reports on the COD reduction from purified terephthalic acid (PTA) wastewater from a petrochemical plant using thermolysis. CuSO4, FeCl3, and FeSO4 were used as chemicals/coagulants for COD reduction. The effect of treatment time, temperature, pH, and chemical dosage on the removal of COD has been studied. FeCl3 was found to be the most effective chemical agent which removed 77.2% COD (from initial COD of 3320 to 765.9 mg/L) at optimum treatment conditions (pH 7, dosage 3 kg/m3 (0.018M), temperature 50 °C, and treatment time 20 min). Temperature played an important role in COD removal. Zeta potential studies depict the change in net surface charge of the particles in all the systems. In the case of CuSO4·5H2O, floc size distribution (number density) shows that bigger flocs were formed at higher temperatures. With iron salts, a decrease in floc size wa...

Binding Affinities of Factor Xa Inhibitors Estimated by Thermodynamic Integration and MM/GBSA

We present free energy estimates of nine 3-amidinobenzyl-1H-indole-2-carboxamide inhibitors of factor Xa. Using alchemical thermodynamic integration (TI) calculations, we estimate the difference in binding free energies with high accuracy and precision, except for mutations involving one of the amidinobenzyl rings. Crystal studies show that the inhibitors may bind in two distinct conformations, and using TI, we show that the two conformations give a similar binding affinity. Furthermore, we show that we can reduce the computational demand, while still retaining a high accuracy and precision, by using fewer integration points and shorter protein-ligand simulations. Finally, we have compared the TI results to those obtained with the simpler MM/GBSA method (molecular-mechanics with generalized Born surface-area solvation). MM/GBSA gives better results for the mutations that involve a change of net charge, but if a precision similar to that of the TI method is required, the MM/GBSA method is actually slightly more expensive. Thus, we have shown that TI could be a valuable tool in drug design.

Membrane surface charge dictates the structure and function of the epithelial Na+/H+exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.

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Release of DNA from polyelectrolyte multilayers fabricated using ‘charge-shifting’ cationic polymers: Tunable temporal control and sequential, multi-agent release

We report an approach to the design of multilayered polyelectrolyte thin films (or ‘polyelectrolyte multilayers’, PEMs) that can be used to provide tunable control over the release of plasmid DNA (or multiple different DNA constructs) from film-coated surfaces. Our approach is based upon methods for the layer-by-layer assembly of DNA-containing thin films, and exploits the properties of a new class of cationic ‘charge-shifting’ polymers (amine functionalized polymers that undergo gradual changes in net charge upon side chain ester hydrolysis) to provide control over the rates at which these films erode and release DNA. We synthesized two ‘charge-shifting’ polymers (polymers 1 and 2) containing different side chain structures by ring-opening reactions of poly(2-alkenyl azlactone)s with two different tertiary amine functionalized alcohols (3-dimethylamino-1-propanol and 2-dimethylaminoethanol, respectively). Subsequent characterization revealed large changes in the rates of side chain ester hydrolysis for these two polymers; whereas the half-life for the hydrolysis of the esters in polymer 1 was ~200days, the half-life for polymer 2 was ~6days. We demonstrate that these large differences in side chain hydrolysis make possible the design of PEMs that erode and promote the surface-mediated release of DNA either rapidly (e.g., over ~3days for films fabricated using polymer 2) or slowly (e.g., over ~1month for films fabricated using polymer 1). We demonstrate further that it is possible to design films with release profiles that are intermediate to these two extremes by fabricating films using solutions containing different mixtures of these two polymers. This approach can thus expand the usefulness of these two polymers and achieve a broader range of DNA release profiles without the need to synthesize polymers with new structures or properties. Finally, we demonstrate that polymers 1 and 2 can be used to fabricate multilayered films with hierarchical structures that promote the sequential release of two different DNA constructs with separate and distinct release profiles (e.g., the release of a first construct over a period of ~3days, followed by the sustained release of a second for a period of ~70days). With further development, this approach could contribute to the design of functional thin films and surface coatings that provide sophisticated control over the timing and the order of the release of two or more DNA constructs (or other agents) of interest in a range of biomedical contexts.

Characterisation of myosin heavy chain gene variants in the fast and slow muscle fibres of gammarid amphipods

Recent molecular work has revealed a large diversity of myosin heavy chain (MyHC) gene variants in the abdominal musculature of gammarid amphipods. An unusual truncated MyHC transcript from the loop 1 region (Variant A 3) was consistently observed in multiple species and populations. The current study aimed to determine whether this MyHC variant is specific to a particular muscle fibre type, as a change in net charge to the loop 1 region of Variant A 3 could be functionally significant. The localisation of different fibre types within the abdominal musculature of several gammarid species revealed that the deep flexor and extensor muscles are fast-twitch muscle fibres. The dorsal superficial muscles were identified as slow fibres and the muscles extrinsic to the pleopods were identified as intermediate fibres. Amplification of loop 1 region mRNA from isolated superficial extensor and deep flexor muscles, and subsequent liquid chromatography and sequence analysis revealed that Variant A 3 was the primary MyHC variant in slow muscles, and the conserved A 1 sequence was the primary variant in fast muscles. The specific role of Variant A 3 in the slow muscles remains to be investigated.

Effects of Protein Microstructure on the Retention Time of T4 Lysozyme Variants in Cation Exchange Chromatography

The structure of a protein directly affects its function. Therefore, characterization of recombinant protein structures is important but is a challenging task. One of the important forces that play a major role in maintaining both structural and functional properties of proteins is electrostatic interactions among different amino acid residues. In this article, cation exchange chromatography was used to study how the microstructure of some charged amino acid residues may affect a protein's retention. Two sets of T4 lysozyme variants were generated. The first set included seven variants that varied in their charge distribution. These variants were obtained by replacing a charged amino acid residue at different sites on lysozyme. The second set included ten variants that varied in both net charge and charge distribution, and these variants were obtained by replacing charged and neutral amino acid residues at different sites on the protein. The microstructure was quantified by calculating the relative hydrophilicity around the replacing amino acid residue. The retention times of all variants were compared with the retention time of the respective control variant. Among the first set of variants, there was a direct correlation (R(2) = 0.93) between the relative hydrophilicity of the replaced amino acid and the protein's retention time, except for two variants (K83H and K124H) whose replacing amino acid residue was involved in intramolecular interactions. For the second set, there was a direct correlation (R(2) = 0.97) between the change in net charge (-2 to +2 units) and the retention times. However, the retention times of two variants (R76D and R76S) did not follow the correlation. We hypothesize that the structure around the replacing charged group is responsible for the deviated protein retention pattern.

Rational Design of Antimicrobial C3a Analogues with Enhanced Effects against Staphylococci Using an Integrated Structure and Function-Based Approach

The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.

Physicochemical properties and microstructures of soy protein isolate gels produced using combined cross-linking treatments of microbial transglutaminase and Maillard cross-linking

Abstract Soy protein isolate (SPI) was incubated with microbial transglutaminase (MTG) enzyme for 5 (SPI/MTG(5)) or 24 (SPI/MTG(24)) h at 40 °C and the gel obtained was freeze-dried, and heated with 2% (w/v) ribose (R) for 2 h at 95 °C to produce combined-treated gels. Control experiments were run with sucrose. Stress relaxation experiments indicated that combined-treated gels were higher in the compressive gel strength, viscoelasticity properties ( K 1 and K 2 ), solidity ( E a ), and were lower in % deformation as compared to single-treated or control gels (gels produced without cross-linking treatment). At most of pHs tested, the solubility of single-treated or control gels were always higher than that of the combined-treated gels. The pH ranges at which the gels were least soluble were wider in the combined-treated gels compared to those of other gels. Field emission scanning electron microscope (FESEM) analysis revealed a denser and finer network of combined-treated gels as compared to that of other gels. The occurrence of covalent cross-links within the network of the gels as well as changes in net charge of soy protein had all affected the physicochemical properties and the microstructure of the combined-treated gels.

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (BA), bacteriopheopytin (HA), the primary quinone (QA) to the secondary quinone (QB). Although the non-heme iron complex (Fe complex) is located between QA and QB, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637–644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459–4465]. In this study, we calculated redox potentials of QA/B (Em(QA/B)) and the Fe complex (Em(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. Em(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic Em(Fe) downshift by 230 mV upon formation of QA− (but not QB−) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.

Large and Giant Vesicles “Decorated” with Chitosan: Effects of pH, Salt or Glucose Stress, and Surface Adhesion

This paper describes the behavior of large and giant unilamellar vesicles (LUVs and GUVs, respectively) in the presence of chitosan, a positively charged polyelectrolyte. Variation of the zeta-potential of LUVs as a function of chitosan concentration is studied for two different molecular weights (MW) after a preliminary study devoted to pH and salt effects on zeta-potential in order to discriminate among the effects of protons, salt, and chitosan concentrations. The difference observed between pH and salt effects on the one hand and chitosan on the other allows us to conclude there is a strong LUV-chitosan interaction. In presence of chitosan, the zeta-potential of LUVs becomes positive and two distinct regimes of variation are suggested and interpreted as follows: a first step consists of chitosan adsorption flat on the membrane (independent of MW) followed by a possible reorganization of the polymer of higher molecular weight on the surface, giving rise to loops. Then a comparative observation of the effect of pH and salt by optical microscopy is made on naked and chitosan-decorated GUVs. Results further confirm a membrane-chitosan interaction and are interpreted in the light of the results obtained for LUVs in terms of both electrostatic and hydrophobic interaction. A large majority of decorated vesicles remain stable down to pH = 1 while in the absence of chitosan they burst quickly at pH between 2 and 3. Osmotic pressure and net charge change due to addition of HCl results in a decrease in the diameter of the decorated vesicles, which remain spherical while forming tubes of lipids. In presence of NaCl, a higher resistance of decorated vesicles is also evidenced (they are stable for NaCl concentrations up to 10-1 M while naked vesicles burst when [NaCl] is between 10-2 and 10-3 M). At higher salt concentration, aggregation of decorated vesicles occurs, which is attributed to the screening of electrostatic repulsions between vesicles covered by the positively charged chitosan. Finally, adhesion of vesicles on a positively charged surface is investigated. In absence of chitosan, the vesicles immediately burst when they come in contact with the surface. On the contrary, suspension of chitosan-vesicles remain stable down to pH = 1.5. Under gentle flow vesicles move: they do not adhere on the substrate, probably due to the repulsion between positively adsorbed charged chitosan and substrate; spherical deflation occurs, but in this case daughter vesicles are formed instead of lipid tubes.

The molecular characterisation and antimicrobial activity of amidated bovine lactoferrin

Chemical modification of bovine lactoferrin (LF) by amidation increased the net positive charge of the protein and markedly enhanced its activity against most Gram-positive and Gram-negative bacteria tested, but not against Saccharomyces cerevisiae or Penicillium candidum. Bovine lactoferrin was amidated with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide in the presence of ammonium ions. Mass spectrometric analysis of the modified protein confirmed that amidation converted aspartyl or glutamyl residues to asparaginyl or glutaminyl residues. The changes in net charge and electrophoretic mobility of amidated LF were confirmed by electrophoretic and chromatographic methods. Isoionic point titration was used to determine changes to net charges and ion exchange chromatography results confirmed that the increased positive charge played an important role in the antimicrobial action of the amidated protein. Thus, chemical amidation of LF introduced substantial structural changes in the LF molecule, reflected in amino acid composition and increased net positive charge, and enhanced biological activity.

Theoretical investigation on the one- and two-photon absorption properties for a series of symmetrical charge transfer fluorene–phenylene and fluorene–thiophene derivatives

One- and two-photon absorption properties of a series of fluorene derivatives with symmetrical charge transfer D–π–D and A–π–A structural motifs have been theoretically investigated with ZINDO/S method. The optimized structures and the characterization of frontier molecular orbitals were obtained by using AM1 calculations. Two-photon absorption properties of molecules have been studied using three-state model. The calculation results have shown that fluorene–thiophene derivatives exhibit larger two-photon absorption cross-section as compared with other studied molecules. To illustrate the results, the crucial effects of thiophene ring on fluorene-thiophene derivatives and the net charge changes on the π-conjugated bridges are analyzed theoretically.