Non-hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single-stranded DNA.

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label-free, and economical non-hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single-stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single-base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84-mer, in which guanine to adenine single-base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.

Safe and rapid development of capillary electrophoresis for ultratrace uranyl ions in radioactive samples by way of fluorescent probe selection for actinide ions from a chemical library.

After the serious nuclear accident at the Fukushima Daiichi Nuclear Power Plant caused by the Great East Japan Earthquake in 2011, the development of feasible, safe, and highly sensitive analytical methods (in terms of low levels of radiation exposure and radioactive waste generation) for radioactive samples, especially actinide (An) ions, represents an important challenge. Here we propose a methodology for selecting appropriate emissive probes for An ions with very low consumption and emission of radioactivity by capillary electrophoresis-laser-induced fluorescence detection (CE-LIF), using a small chemical library of probes with eight different chelating moieties. It was found that the emissive probe L1, which possesses the tetradentate chelating moiety 1,10-phenanthroline-2,9-dicarboxylic acid (PDA), was suitable for detecting uranyl ions. The detection limit for the uranyl-L1 complex using CE-LIF combined with dynamic ternary complexation and on-capillary concentration techniques was determined to be 2.9 × 10-12 M (0.7 ppt). No interference from the large excess of matrix metal ions was observed. This method was successfully applied to real radioactive liquid samples collected from nuclear facilities, including the Fukushima Daiichi Nuclear Power Plant. This strategy not only permitted the development of a safe and rapid analytical method but also provided insight into the coordination chemistry of An ion complexes. Specifically, the PDA structure provided substantial kinetic inertness to its uranyl complex; the formation of a ternary complex between uranyl-L1 and carbonate was revealed; and unusual interactions were observed between the π-electron systems of uranyl and the phenanthroline ring, which stabilized the uranyl-PDA interaction.

Separation of single-base sequential isomers of single-stranded DNA by capillary electrophoresis and its application in the discrimination of single-base DNA mutations.

Separation of single-base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single-base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single-base sequential isomers of 12-mer single-stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68-mer gene fragment and its single-base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single-base gene mutations.

High kinetic stability of Zn(II) coordinated by the tris(histidine) unit of carbonic anhydrase towards solvolytic dissociation studied by affinity capillary electrophoresis.

Solvolytic dissociation rate constants (kd) of bovine carbonic anhydrase II (CA) and its metallovariants (M-CAs, M=Co(II), Ni(II), Cu(II), Zn(II), and Cd(II)) were estimated by a ligand substitution reaction, which was monitored by affinity capillary electrophoresis to selectively detect the undissociated CAs in the reaction mixture. Using EDTA as the competing ligand for Zn-CA, the dissociation followed the unimolecular nucleophilic substitution (SN1) mechanism with kd=1.0×10(-7)s(-1) (pH7.4, 25°C). The corresponding solvolysis half-life (t1/2) was 80days, showing the exceptionally high kinetic stability of t Zn-CA, in contrast to the highly labile [Zn(II)(H2O)6](2+), where the water exchange rate (kex) is high. This behavior is attributed to the tetrahedral coordination geometry supported by the tris(histidine) unit (His3) of CA. In the case of Co-CA, it showed a somewhat larger kd value (5.7×10(-7)s(-1), pH7.4, 25°C) even though it shares the same tetrahedral coordination environment with Zn-CA, suggesting that the d(7) electronic configuration of Co(II) in the transition state of the dissociation is stabilized by the ligand field. Among M-CAs, only Ni-CA showed a bimolecular nucleophilic substitution (SN2) reaction path in its reaction with EDTA, implying that the large coordination number (6) of Ni(II) in Ni-CA allows EDTA to form an EDTA-Ni-CA intermediate. Overall, kd values roughly correlated with kex values among M-CAs, with the kd value of Zn-CA deviating strongly from the trend and highlighting the exceptionally high kinetic stabilization of Zn-CA by the His3 unit.

Thermodynamics of binding of a sulfonamide inhibitor to metal-mutated carbonic anhydrase as studied by affinity capillary electrophoresis.

By affinity capillary electrophoresis (ACE), the thermodynamic binding constants of a sulfonamide (SA) inhibitor to bovine carbonic anhydrase II (CA) and metal mutated variants (M-CAs) were evaluated. 1-(4-Aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate was used as the SA in the electrophoretic buffer for ACE. The Scatchard analysis of the dependence of the electrophoretic mobility of native CA on the SA concentration provided the binding constant to be Kb=(2.29±0.05)×10(6) M(-1) (at pH8.4, 25°C). On the other hand, apoCA showed far smaller value [Kb=(3.76±0.14)×10(2) M(-1)], suggesting that the coordination of SA to the Zn(II) center controlled the binding thermodynamics. The ACE of M-CAs showed the same behaviors as native CA but with different Kb values. For example, Co-CA adopting the same tetrahedral coordination geometry as native CA exhibited the largest Kb value [(2.55±0.05)×10(6) M(-1)] among the M-CAs. In contrast, Mn- and Ni-CA, which adopted the octahedral coordination geometry, had Kb values that were about two orders of magnitude lower. Because the hydrophobic cavity of CA around the active center pre-organized the orientation of SA, thereby fixing the ligating NH(-) moiety to the apex of the tetrahedron supported by three basal His3 of CA, metals such as Zn and Co at the center of M-CA gave the most stable CA-SA complex. However, pre-organization was not favored for octahedral geometry. Thus, pre-organization of SA was the key to facilitating the tetrahedral coordination geometry of the Zn(II) active center of CA.

Application of capillary electrophoresis with laser-induced fluorescence detection for the determination of trace neodymium in spent nuclear fuel using complexation with an emissive macrocyclic polyaminocarboxylate probe.

A simple and rapid method with low radiation exposure risk was developed for the determination of neodymium in spent nuclear fuel by capillary electrophoresis with laser-induced fluorescence detection using a fluorescent probe having a macrocyclic hexadentate polyaminocarboxylate structure. The concentration of Nd(III) in a spent nuclear fuel sample was determined with no interference from various matrix elements, including lanthanides and uranium (at a 200-fold excess), with 92 ± 3% recovery. This is due to high resolution based on establishing a ternary complex equilibrium during migration in which the hydroxyl ion plays an auxiliary role (log K(Ln-L-OH) = 3.9-5.3).

Dynamic coating capillary electrophoresis for separation of humic acid using mixture solution of non-ionic polymers both as coating agent and separation medium.

A simple, rapid, and precise dynamic coating capillary electrophoretic separation method for water-soluble humic substances is proposed. An aqueous solution containing hydroxyethyl cellulose (HEC), polyethylene glycol (PEG), and pH buffer component was employed for both the dynamic coating agent and the separation medium. The procedure for the coating of the capillary inner wall was simply filling the buffered polymer mixture solution into the capillary that had been treated with 1 M aqueous HCl solution. The solution for the capillary coating was directly used as the electrophoretic buffer solution for CE separation. Excellent performance for the separation of humic acid was obtained using the solution containing 0.5% (w/v) HEC, 1.0% (w/v) PEG 10000, and 0.1% (w/v) PEG 8000000. Excellent reproducibility and durability were obtained even at slightly alkaline conditions at pH levels above 8. The separation of 0.1 - 2 kbp of DNA ladder by the proposed method showed was also achieved.

Dissociation kinetic analysis of Ce(III) complex with Quin2 by microchip capillary electrophoretic reactor.

Dissociation kinetic analysis of a complex of Ce(3+) with a polyaminocarboxylic ligand, 8-amino-2-[(2-amino-5-methylphenoxyl)methyl]-6-methoxyquinoline-N,N,N',N',-tetraacetic acid (Quin2), was studied by microchip capillary electrophoretic reactor. Dissociation rate constants, k(d), of Ce(3+)-Quin2 complex in alkaline conditions at pH 8.3 - 9.8 were determined. The linear relationship of k(d) with the concentration of hydroxide ion indicates the existence of a hydroxide ion-assisted path in the dissociation reaction of Ce(3+)-Quin2 complex in alkaline conditions. The solvolytic dissociation rate constant, and the hydroxide ion-assisted dissociation rate constant of Ce(3+)-Quin2 complex were determined to be 1.55 × 10(-3) and 3.24 × 10(2) s(-1) in the analysis of the dependence of k(d) with the concentration of hydroxide ion, respectively.

A molecular probe for recognizing the size of hydrophobic cavities based on near-infrared absorbing diradical-Pt(II) complexes.

A diradical-platinum(II) complex was able to recognize the subtle difference in cavity size between ß- and γ-cyclodextrin with on-off switching of intense near-infrared absorption. This provides a new probe for identifying the size of hydrophobic cavities, which has been successfully applied here to differentiate human serum albumin from α-chymotrypsin.

Computerized surveillance system of tuberculosis in Japan: its evolution, achievement and challenges.

The nationwide computerized tuberculosis (TB) surveillance system was revised in 2007. It was developed to be user-friendly and to allow the evaluation of current TB problems and control issues in Japan. All public health centers in Japan (518 as of April 2007) have system terminals connected to a central computer, and the data entered at these terminals are sent to the online central computer excluding personal identification data. All the figures and tables in this paper were created using the annual report database which are compiled from this system. The revision in 2007 added many new functions to the system, such as a function for automatically sending data upon transfer. The monitoring information for assisting case management of TB patients by the DOTS was also enhanced. The algorithm for classifying treatment outcomes automatically based on data entered regarding cancellations from registration, bacteriological results and drug usage each month was revised. The proportion of "Failed" and "Defaulted" combined was 4.6% among new sputum smear positive pulmonary TB patients newly registered in 2009, while "Died" accounted for as high as 19.3%, due largely to a high percentage of the elderly. A new system for contact examination management is provided as a subsystem. Feedback of data analyses has been strengthened by various methods. This TB surveillance system is indispensable for implementing the evidence-based TB control program in Japan. An important role of the Research Institute of Tuberculosis is to support the planning and execution of TB control with provision of useful epidemiological information from the system.

Highly efficient near-infrared-emitting lanthanide(III) complexes formed by heterogeneous self-assembly of Ag(I), Ln(III), and thiacalix[4]arene-p-tetrasulfonate in aqueous solution (Ln(III) = Nd(III), Yb(III)).

Heterogeneous self-assembly of thiacalix[4]arene-p-tetrasulfonate (TCAS), Ag(I), and Ln(III) (= Nd(III), Yb(III)) in aqueous solutions conveniently afforded ternary complexes emitting Ln(III)-centered luminescence in the near-infrared (NIR) region. A solution-state study revealed that the Ag(I)-Nd(III)-TCAS system gave a complex Ag(I)(4)·Nd(III)·TCAS(2) in a wide pH range of 6-12. In contrast, the Ag(I)-Yb(III)-TCAS system gave Ag(I)(2)·Yb(III)(2)·TCAS(2) at a pH of around 6 and Ag(I)(2)·Yb(III)·TCAS(2) at a pH of approximately 9.5. The structures of the Yb(III) complexes were proposed based on comparison with known Ag(I)-Tb(III)-TCAS complexes that show the same self-assembly behavior. In Ag(I)(2)·Yb(III)(2)·TCAS(2), two TCAS ligands sandwiched a cyclic array of a Ag(I)-Ag(I)-Yb(III)-Yb(III) core. In Ag(I)(2)·Yb(III)·TCAS(2), Yb(III) was accommodated in an O(8) cube consisting of eight phenolate O(-) groups from two TCAS ligands linked by two S-Ag-S linkages. Crystallographic analysis of Ag(I)(4)·Nd(III)·TCAS(2) revealed that the structure was similar to Ag(I)(2)·Yb(III)·TCAS(2) but that it had four instead of two S-Ag-S linkages. The number of water molecules coordinating to Ln(III) (q) estimated on the basis of the luminescent lifetimes was as follows: Ag(I)(4)·Nd(III)·TCAS(2), 0; Ag(I)(2)·Yb(III)(2)·TCAS(2), 2.4; and Ag(I)(2)·Yb(III)·TCAS(2), 0. These findings were compatible with the solution-state structures. The luminescent quantum yield (Φ) for Ag(I)(4)·Nd(III)·TCAS(2) was 4.9 × 10(-4), which is the second largest value ever reported in H(2)O. These findings suggest that the O(8) cube is an ideal environment to circumvent deactivation via O-H oscillation of coordinating water. The Φ values for Ag(I)(2)·Yb(III)(2)·TCAS(2) and Ag(I)(2)·Yb(III)·TCAS(2) were found to be 3.8 × 10(-4) and 3.3 × 10(-3), respectively, reflecting the q value. Overall, these results indicate that the ternary systems have the potential for a noncovalent strategy via self-assembly of the multidentate ligand, Ln(III), and an auxiliary metal ion to obtain a highly efficient NIR-emissive Ln(III) complex that usually relies on elaborate covalent linkage of a chromophore and multidentate ligands to expel coordinating water.

[Present activity of BCG and LTBI treatment for infants in Japan].

I analyzed the BCG vaccination and treatment of latent tuberculosis infection (LTBI) for infants notified in 2008 in Japan. BCG was not recommended and treatment of LTBI was the main activity for prevention of TB in infants from birth to age 2 months. The majority of notified LTBI cases were detected by contact surveys. Out of the estimated number of TB infected (148), only 2 cases were notified based on the outcome of LTBI treatment for 89 infants. When the infants were 3-5 months old, both BCG vaccination and LTBI treatment were implemented. BCG coverage was 61.5-97.7%, and LTBI treatment for non-vaccinated individuals was applied for a larger number of infants (1.04 to 7.82 times as many) than the estimated number of infants infected with TB. The majority of infants were BCG vaccinated when they were 6-11 months old. Although LTBI treatment coverage was low, only 5 cases developed among those receiving BCG vaccination. During 1-2 year, BCG coverage was high and breakdown rates of BCG-vaccinated children were much lower than those of non-vaccinated children. This difference might be due to not only the preventive effect of BCG but also risk difference of TB infection between BCG-vaccinated and BCG non-vaccinated individuals. The number of notified LTBI treatment cases was lower than the estimated number of children infected with TB during 1-2 year. To prevent infant TB, reinforcement of contact surveys to identify LTBI for treatment, improvement of BCG coverage, and attention to BCG non-vaccinated infants older than 6 months might be effective.

Effect of urea on the ion-association capillary electrophoresis separation of the anionic metal complexes using hydrophobic tetraalkylammonium as ion-association agent.

The effect of urea as an electrophoretic buffer solution modifier on the ion-association (IA) capillary electrophoresis (CE) separation of four anionic metal complexes of Al(III), Co(III), Cr(III), and Fe(III) with 2,2'-dihydroxyazobenezene-5,5'-disulfonate (DHABS) using a hydrophobic ion-association agent, tetrapentylammonium bromide, was studied. The mutual separation of the four anionic metal-DHABS complexes was not achieved without the addition of urea in the electrophoretic buffer solution. However, the addition of 1.5 M urea in the electrophoretic buffer solution brought about a complete separation of the four metal complexes. The ion-association constants between all metal-DHABS complexes and tetrapentylammonium in an aqueous urea solution were smaller than those in a neat aqueous solution. This indicates the hydrophobic interaction contributing to the ion-association between analytes and ion-association agent during IA-CE separation processes can be controlled with the addition of urea to the electrophoretic buffer solution. Another advantage of adding urea was a substantial enhancement of separation efficiency with a reduction of the half-bandwidth of the peaks. Also, a reduction of the electrophoretic mobility of the electroosmotic flow when urea was added was much less than when organic solvents were used.

[Trends in tuberculosis infection among foreigners in Japan according to work status].

PURPOSE: TB among foreigners is presently a serious issue in some developed countries and could become so in Japan. The purpose of this report is to assess the epidemiological situation of TB among foreigners in Japan. MATERIALS AND METHODS: The trend of TB reporting among foreigners in Japan was examined with regard to work status. RESULTS: The number of reported TB cases among employees and students in Japan increased between 1998 and 2008, but that among housekeepers was level throughout the same period. The increase among employees and students might be due to the increased numbers of foreign employees and students. In the case of housekeepers, the increase in the number of permanent residents did not lead to an increase in TB among these housekeepers. Estimates of TB reporting rates decreased during the study period, so the changes in reporting rates would not have caused the increase in TB cases. This downward trend may have been caused by an increase in longer-term residents and a decrease in TB incidence in home countries. Even though the TB reporting rate is decreasing, the rates in those countries are much higher than in Japan in the same work categories. DISCUSSION: To control the spread of TB, it is important to identify high-risk individuals. The Japanese TB control program should further strengthen mass health examination programs for foreign housekeepers and employees (especially temporary and daily employees), case-finding based on individuals' access to hospitals or clinics when suffering from TB symptoms, and flexible and periodic adjustment of TB control activities for foreigners according to future changes in the number and distribution of foreigners in Japan. Furthermore, improving the TB epidemiological situation in home countries might contribute to the downward trend of TB reporting rates among foreigners in Japan. Therefore, Japanese assistance in TB control activities in surrounding countries such as China, South Korea, and the Philippines might contribute to TB control activities for foreigners in Japan.

[Recent case-finding activities of school children in Japan].

PURPOSE: The procedure for TB mass-screening of elementary and junior-high school students was revised in 2003 in Japan. An interview sheet with questions about the past history of TB, episodes of contact with TB patient, TB symptoms, and BCG vaccination history has been administered to all students to determine high-risk groups for TB infection or TB disease despite previous mass-screening with PPD testing for those in first grade. Our purpose in the present study was to evaluate the effects of revised case-finding activity for these school students. METHOD: We analyzed the case-finding activities for school students from 2003-2008 based on data from the national TB surveillance system and related ad-hoc surveys. RESULT: The total number of notified TB cases among these students was 295 out of which 156 were detected by contact survey, 110 by consultation at medical facilities with symptoms, 19 by mass-screening at schools, and the remaining 10 by other means. Although the contact investigation detected more than half of all cases, there were some more cases with a known source of infection among those who were detected in other modes; 28 cases were identified by consultation at medical facilities and 10 by school mass-screening. Case-finding activities by consultation at medical facilities detected 43 bacteriologically positive cases. There were 97 cases without a known source of infection. Most of the cases detected by school mass-examination had risk factors such as TB history in the family and/or a history of staying in TB-prevalent countries. The TB incidence in urban areas is higher than that in other areas for junior-high school students with known or unknown sources of infection. DISCUSSION: The effectiveness of the contact investigation could be enhanced by improving its way of implementation. Delay of diagnosis for infectious cases should be minimized as much as possible to prevent TB outbreaks among students. For that purpose, parents, school teachers, and school nurses should be aware to prevent a delay in seeking care for the patient. Because the efficiency of the current school mass-screening is quite low, revision of this program is necessary, taking into consideration the reinforcement of other case-finding activities. The difference between urban and other areas might be due to a higher infection risk in junior-high school students in urban areas who have higher social activity and may be exposed to higher TB infection risk due to casual contact.

[Influence of occupation of the index cases in recent outbreaks of tuberculosis].

UNLABELLED: PURPOSE, MATERIALS AND METHODS: In order to elucidate risk factors of TB outbreaks, 223 outbreaks reported between 2000 and 2006 in Japan were analyzed according to sex, occupation and outbreaking place of the index case. RESULTS: In male, the most frequent occupation was permanent workers who infected contacts mainly at workplaces, followed by unemployed people at general hospital or nursing home for the aged. The third was students who infected contacts mostly at school, followed by customer relations workers at companies or amusement places. In female, the most frequent occupation was students who infected contacts at school. Second most frequent was unemployed who infected contacts at general hospitals and nursing homes for the aged, followed by nurses at general hospitals or mental hospitals. Frequent outbreak places were workplaces, schools, general hospitals, amusement places and mental hospitals in male, and general hospitals and schools in female. The risk of becoming the source of TB outbreak was high in teachers/doctors, students, unemployed as well as customer relations workers in male, and teachers/doctors, students as well as nurses in female. DISCUSSION: The most important point to prevent outbreaks of TB is to find out TB suspects with relevant symptoms for tuberculosis as early as possible to minimize patient's delay, especially from people with high risk of TB infection to others such as teachers/doctors, students and medical nurses. As most frequent job and place were general permanent workers at workplaces, TB control activities to minimize patient's delay at general workplace is also important.

[A case of multiple-drug-resistant pulmonary tuberculosis in a hemodialysis hospital].

A 42-year-old male with chronic renal failure was diagnosed as multiple-drug-resistant pulmonary tuberculosis, 9 months after the onset of hemodialysis. During the period before the diagnosis of tuberculosis, he visited regularly the hemodialysis hospital, accordingly many patients with hemodialysis and the hospital staffs had close and long contact with the patient. Our health center planned and conducted contact examinations among them, with QFT-TB test and chest X-ray. Petients with hemodialysis are regarded as immunocompromised hosts, one of the high risk groups for infections. The result of QFT-TB test of patients were negative, however, we have to cosider the possibility of false-negative. So we followed up by monthly chest X-ray examination all hemodialysis patients for 2 years, and finally, it was found that secondary infection of multiple-drug-resistant tuberculosis among immunocompromised hosts did not occur.

Detection of cationic guest molecules by quenching of luminescence of a self-assembled host molecule consisting of terbium(III) and calix[4]arene-p-tetrasulfonates.

By self-assembly in aqueous solution, calix- (CAS) and thiacalix[4]arene-p-tetrasulfonate (TCAS) formed luminescent complexes Tb(III).(CAS)2 and Tb(III).TCAS, respectively, which were utilized as a host for cationic guests. Addition of 1-ethylpyridinium guest quenched luminescence of Tb(III).(CAS)2 in accordance with the Stern-Volmer (SV) relation with a low detection limit (D.L.) of 5.94 x 10(-8) M (S/N=3, M identical with mol dm(-3)). On the other hand, 1-ethylquinolinium quenched luminescence of Tb(III).TCAS most efficiently, affording a very low D.L. (6.71 x 10(-10) M). The agreement of the SV coefficients obtained with luminescent intensity (K(SV,all)=6.74 x 10(6) M(-1)) and lifetime (K(SV,Tb)=6.50 x 10(6) M(-1)) implied that dynamic quenching of 5D4 excited state of Tb(III) was predominant in the quenching processes. The quenching rate was estimated to be k(q,Tb)=9.94 x 10(9) M(-1) s(-1), which was as fast as diffusion-limited rate. Quenching of Tb(III).(CAS)2 was also applied to detection of NAD+, with a D.L. of 2.78 x 10(-7) M.

Expanding the scope of CE reactor to ssDNA-binding protein-ssDNA complexes as exemplified for a tool for direct measurement of dissociation kinetics of biomolecular complexes.

CE reactor (CER), which was developed as a tool for direct measurement of the dissociation kinetics of metal complexes, was successfully applied to the complexes of Escherichia coli ssDNA-binding protein (SSB) with ssDNA. The basic concept of CER is the application of CE separation process as a dissociation kinetic reactor for the complex, and the observation of the on-capillary dissociation reaction profile of the complex as the decrease of the peak height of the complex with increase of the migration time. The peak height of [SSB-ssDNA] decreases as the migration time increases since the degree of the decrease of [SSB-ssDNA] through the on-capillary dissociation reaction is proportional to the degree of the decrease of the peak height of [SSB-ssDNA]. The dissociation degree-time profiles for the complexes are quantitatively described by analyzing a set of electropherograms with different migration times. Dissociation rate constants of [SSB-ssDNA] consisting of 20-mer, 25-mer and 31-mer ssDNA were directly determined to be 3.99x10(-4), 4.82x10(-4) and 1.50x10(-3)/s, respectively. CER is a concise and effective tool for dissociation kinetic analysis of biomolecular complexes.

Ligand-substitution mode capillary electrophoretic reactor: extending capillary electrophoretic reactor toward measurement of slow dissociation kinetics with a half-life of hours.

A method employing capillary electrophoresis (CE) was developed to determine the rate constant of the very slow spontaneous dissociation of a complex species. The method uses a CE reactor (CER) to electrophoretically separate components from a complex zone and, thus, spontaneously dissociate a complex. The dissociation is accelerated by ligand substitution (LS) involving a competing ligand added to the electrophoretic buffer. The LS-CER method is validated using the dissociation of a Ti(IV)-catechin complex and EDTA as a competing ligand. There is good agreement between the spontaneous dissociation rate constant (k(d) = (1.64 +/- 0.63) x 10(-4) s(-1)) and the rate constant obtained by a conventional batchwise LS reaction (k(d) = (1.43 +/- 0.04) x 10(-4) s(-1)). Furthermore, the usefulness of the method is demonstrated using a Ti(IV)-tiron complex, for which k(d) = (0.51 +/- 0.43) x 10(-4) s(-1), corresponding to a half-life (t(1/2)) of 3.8 h. Notably, a single run of LS-CER for the Ti(IV) complex is completed within 40 min, implying that LS-CER requires a considerably shorter measurement time (roughly equal to t(1/2)) than conventional CER. LS-CER can be widely applied to determine the spontaneous dissociation rates of inorganic diagnostic and therapeutic reagents as well as of biomolecular complexes.