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

Research Article

Expanding the scope of CE reactor tossDNA-binding protein–ssDNA complexesas exemplified for a tool for directmeasurement of dissociation kinetics ofbiomolecular complexes

CE reactor (CER), which was developed as a tool for direct measurement of the disso-

ciation 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 3.99� 10�4, 4.82� 10�4 and 1.50� 10�3/s, respectively. CER is a concise and

effective tool for dissociation kinetic analysis of biomolecular complexes.

Keywords:

Biomolecular complex / CE / Dissociation kinetic analysisDOI 10.1002/elps.200900110

1 Introduction

There is no question as to the importance of biomolecular

interaction analysis, which is one of the molecular biological

approaches in the understanding of the life phenomenon.

As for the research trends in biomolecular interaction

analysis, structural or thermodynamic approaches are the

mainstream. Consideration about the dynamics of biomo-

lecular complexes is, however, indispensable in under-

standing their biological meaning because biomolecules are

constantly in flux; the temporary interactions between

biomolecules or their complexes with noncovalent bonding

play important roles in the vital activity in the living cells [1].

In addition, kinetics provide intrinsic information about

reaction mechanisms [2]. There are not a lot of works on

kinetics of biomolecular complex in spite of its widely

recognized importance. This is partly due to the lack of the

convenient methodologies for investigating the kinetics of

the biomolecular complexes. Basically, direct measurement

of their rate constants is technically difficult because of

extreme difficulty in creating a reaction system, where the

reverse-reaction to a reaction of interest does not take place,

in homogeneous solution. In many cases, one, therefore,

could not help measuring them indirectly after repeating

many complex operations in equilibrium state [3, 4]. On the

other hand, the technique using surface plasmon resonance

(SPR) [5] enabled the direct analysis of biomolecular

interaction. It, however, needs immobilization of the target

molecules on the gold thin film surface of the flow-cell with

tangled procedures. The deactivation of the target molecules

arising from immobilization is also concerned. In addition,

the transport of the analyte between the immobilized

surface- and the liquid-phase complicates the handling of

the kinetic data.

Recently, a review was published, where CE is a

promising technique for the investigation of molecular

interactions especially in obtaining kinetic information [6].

We also had exemplified the advantages in CE for kinetic

Toru TakahashiKei-ichirou OhtsukaYoriyuki TomiyaNobuhiko IkiHitoshi Hoshino

Division of EnvironmentallyBenign Systems, GraduateSchool of Environmental Studies,Tohoku University, Sendai,Japan

Received February 23, 2009Revised April 23, 2009Accepted May 12, 2009

Abbreviations: CER, CE reactor; lCER, microchip CEreactor; SPR, surface plasmon resonance; SSB, SSB-binding protein; TPP1, tetraphenylphosphonium; TPPCl,

tetraphenylphosphonium chloride

Correspondence: Dr. Toru Takahashi, Division of Environmen-tally Benign Systems, Graduate School of EnvironmentalStudies, Tohoku University, 20 Aoba, Aramaki, Aoba-ku, Sendai980-8579, JapanE-mail: [email protected]: 181-22-795-7223

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 3079–3085 3079

studies by a report in which a unique approach for the

dissociation kinetic measurement of metal complexes viaCE reactor (CER) [7] was described well before the review

was issued. Chemical basis of CER is just based on the fact

observing the on-capillary dissociation reaction profile of

the metal complexes in the pre-capillary derivatization

CE employing an electrophoretic buffer solution without

chelating reagent [7, 8]. In above CE system, metal

complexes, which migrate in their isolated bands along a

capillary from those of free ligand and metal ions, are

exposed to an overwhelming force causing dissociation by

CE resolution because of the absence of the free ligand

in the electrophoretic buffer solution. One can obtain the

dissociation rate constant (kd) of the metal complex by

analyzing the first-order decay dissociation degree-time

profile directly, readily and accurately. An approach by

CE-based dissociation kinetic analysis methods [7–10]

is the sole one, which enables the direct measurement

of kd of metal complex in homogeneous solution because

CE is a one-of-a-kind method which can create the driving

force for the dissociation reaction of metal complex in

homogeneous solution. Another advantage of CER is that

the estimation of the dissociation reaction profile of the

complex is accurate because of employing the degree of

decrease of the peak height of the complex with increase

of the migration time for the estimation of the degree of

the decomposition of the complex. The estimation of the

degree of the decomposition of the complex by the ratio of

the peak area [11] or the direct fit to the asymmetrical part

of electropherogram [12] may give misguided evaluation

of kd since the boundary between the zone of the complex

and that of the decomposed products is not clear in the

capillary.

It is expected that CER theoretically can be applied to

the dissociation kinetic analysis of complexes in the broad

sense, which are the aggregates of the molecules that are not

bound with covalent bond. In this study, we tried to enhance

the concept of CER to biomolecular complexes. Dissociation

reaction of a protein–DNA complex was chosen as a model

system for kinetic analysis by CER. In the replication of

DNA, ssDNA-binding protein (SSB) stabilizes ssDNA

through binding with the region consisting of about 30

origonucreotides on the template ssDNA chain [13]. As SSB

repeats binding and dissociation with ssDNA according to

the progress of the polymerization of the replicated DNA

chain, dissociation kinetics of the complex of SSB with

ssDNA ([SSB-ssDNA]) has biological importance. In this

study, it has been successfully demonstrated that CE serves

as a chemical reactor for the measurements of dissociation

kinetics of the dissociation kinetics of [SSB-ssDNA]

consisting of Escherichia coli (E. Coli) and ssDNA of 20-mer,

25-mer, and 31-mer. Also such attractive features of CE as

the plug-flow profile, no interactions with a secondary

phase, and the parallel scheme of dissociation and separa-

tion allow the simple treatment of data in the CER. CER is a

concise and effective tool for dissociation kinetic analysis of

not only metal complexes but also biomolecular complexes.

2 Materials and methods

2.1 Reagent and apparatus

An E. coli origin SSB (72 kDa) was purchased from Promega

(Madison, WI, USA). Synthesized ssDNA, 50-cctgccacgctc-

cgctggtt-30 (s20), 50-cctgccacgctccgctggttggtgt-30 (s25) and

50-cctgccacgctccgctggttggtgtggttgg-30 (s31), were purchased

from Nippon EGT (Toyama, Japan). A transferrin

(Tf, 79 kDa) was purchased from Sigma (St. Louis, MO,

USA). A tetraphenylphosphonium chloride (TPPCl) was

purchased from TCI (Tokyo, Japan). All other reagents used

were of guaranteed reagent grade. A laboratory made CE

setup was consisted with a JASCO (Tokyo, Japan) CE-2070

UV-VIS spectrophotometric detector, a Matsusada Precision

(Kusatsu, Japan) HPCZE-30 PO.25 LD high-voltage power

supply, and a Shimadzu (Kyoto, Japan) C-R5A CHROMA-

TOPAC recorder. A GL Science (Tokyo, Japan) fused silica

capillary (50 mm id, total length; 54.5 cm) was used.

Spectrophotometric detection was performed at 200 nm on

the detection window setup from the cathodic side of the

capillary to 12.5 cm. The temperature of the system was kept

at 303 K in a safety box with an interlock system.

2.2 Procedure for dissociation kinetic analysis of

[SSB-ssDNA] by CER

Electrophoresis was performed by constant voltage opera-

tion mode. Before every run, the capillary was conditioned

with 0.1 M NaOH, followed by doubly distilled water and

electrophoretic buffer solution. An electrophoretic buffer

solution containing 15 mM phosphate buffer (pH 7.2) was

used. The sample solution containing 0.12 mg/mL SSB,

4.55 mg/mL ssDNA, 10 mM phosphate buffer (pH 7.2) and

0.25 mM TPPCl is introduced hydrostatically by elevating

the sample at 5 cm for 10 s at anodic side of the capillary.

Several CE experiments with a variety of migration times

while controlling the applied voltage from 5 to 20 kV were

performed. Then, the migration times and the peak height

of [SSB-ssDNA] and tetraphenylphosphonium (TPP1) for

each of electropherograms were recorded. The same

procedure is repeated for the sample containing 0.18

mg/mL Tf, 10 mM phosphate buffer (pH 7.2) and

0.25 mM TPPCl.

3 Results and discussion

3.1 CE separation and the composition of [SSB-

ssDNA]

CE separation of SSB, ssDNA and [SSB-ssDNA] was

examined before CER experiments because dissociation

kinetic analysis of [SSB-ssDNA] with CER would not be

approved if there were overlapping of the peak of SSB

or ssDNA with [SSB-ssDNA]. A single peak except for that of

Electrophoresis 2009, 30, 3079–30853080 T. Takahashi et al.


TPP1 was observed respectively in the electropherogram of

the sample containing only SSB or ssDNA (s31) as shown

in Figs. 1A and C. In this work, because of using an

electrophoretic buffer solution with no addition of SSB or

ssDNA (s31) that composes the complex, the peak of the

complex that is kinetically labile is not observed [8].

However, a new peak of [SSB-ssDNA] completely separated

from that of SSB was observed in the electropherogram of

the sample containing both SSB and ssDNA (s31) as shown

in Fig. 1B. This indicates that [SSB-ssDNA] is kinetically

stable in the time scale of CE separation. In other words,

dissociation reaction of [SSB-ssDNA] is slow enough to

measure its dissociation rate constant by CER. Actually, a

kind of asymmetrical peak shape, i.e. fronting shape, of the

complex peak shows that the complex has dissociated

partially in the CE separation process. The complex

continuously dissociate while migrating in the CE separa-

tion process employing the electrophoretic buffer solution

containing no constituents of the complex. The products of

the dissociation reaction of the complex, i.e. free SSB and

free ssDNA, were immediately separated from the complex

since their electrophoretic mobilities were different. Then,

the products appear as the fronting or the tailing region of

the complex peak. In this case, free SSB particularly

appeared as the fronting part of the complex peak because

of larger apparent electrophoretic mobility of free SSB than

that of the complex.

The composition of [SSB-ssDNA] was also estimated by

CE experiments. It is well known that SSB can bind ssDNA

in various binding modes, referred as (SSB)n, which differ in

the number of nucleotides (n) occluded per bound one SSB,

and that there are two major binding modes, (SSB)35 and

(SSB)65, occlude n 5 3573 and n 5 6573 nucleotides per

SSB [14, 15]. In addition, these two modes quite depend on

the salt concentration [15]. A low salt concentration mode,

(SSB)35, dominates in the present experimental conditions.

Formation of the 1:1 complex is predicted in consideration

of the number of binding positions of SSB at (SSB)35. With

addition of excess SSB to ssDNA (s31), there is no peak

other than free SSB and [SSB-ssDNA] as shown in Fig. 1B.

This strongly suggests that the SSB-ssDNA complexes with

lower molar ratio of ssDNA to SSB than 1 did not form, and

that SSB form the 1:1 complex with ssDNA with addition of

excess SSB to ssDNA. Contrastingly, as shown in the elec-

tropherograms in Fig. 2, with addition of excess ssDNA

(s31) to SSB in the sample, some new broad peaks (pointed

by arrows in the electropherograms) came out behind the

[SSB-ssDNA] peak. The peak height of [SSB-ssDNA] in

Fig. 2A was much smaller than that of in Fig. 1B, in addi-

tion [SSB-ssDNA] peak disappeared in Figs. 2B

and C. This shows the complexes with higher molar ratio of

ssDNA to SSB than 1 dominates with addition of excess

ssDNA to SSB. The migration times of these complexes are

larger than that of 1:1 complex since they have larger

negative charge. Hence, these new peaks are corresponding

to those of these complexes. The results of experiments

do not contradict assumption that the composition of

[SSB-ssDNA] is 1:1. In this study, to avoid formation of

SSB-ssDNA complexes with higher molar ratio, which make

the kinetic analysis of [SSB-ssDNA] complicated, excess SSB

to ssDNA was added in the sample solution.

3.2 Dissociation kinetic analysis of [SSB–ssDNA] by

CER

Figure 3 shows the series of electropherograms of [SSB-

ssDNA] of 20-mer, 25-mer and 31-mer ssDNA with different

0

Migration time / min

TPP+SSB

ssDNATPP+

[SSB-ssDNA]

SSBTPP+

A

B

C

4 8 12

Figure 1. Typical electropherograms of SSB (A), [SSB-ssDNA](B), and ssDNA (C). Sample: 0.12 mg/ml SSB (a), 0.12 mg/ml SSBand 4.55 mg/ml ssDNA (b), and 4.55 mg/ml ssDNA (c). All thesamples contain 10 mM phosphate buffer (pH 7.2) and 0.25 mMTPPCl. Capillary: L 5 42.5 cm, l 5 12.5 cm. Applied voltage: 15 kV.Electrophoretic buffer: 15 mM phosphate (pH 7.2).

0Migration time / min

ssDNA

TPP

TPP

TPP

[SSB-ssDNA]

ssDNA

A

B

C

4 8 12

Figure 2. Typical electropherograms of the complexes of SSBwith ssDNA with different compositions. Sample: 0.12 mg/mlSSB and 9.1 mg/ml ssDNA (A), 0.12 mg/ml SSB and 18.2 mg/mlssDNA (B), 0.12 mg/ml SSB and 36.4 mg/ml ssDNA (C). Otherconditions were the same as Fig. 1.

Electrophoresis 2009, 30, 3079–3085 CE and CEC 3081


migration times obtained with varying applied voltages. The

electrophoretic buffer containing no constituents of the

complex, such as SSB and ssDNA, was used. As shown in

Fig. 3A, the degree of decrease of the peak height of the

complex of SSB with s31 ([SSB-s31]) to the increase of

migration time is much larger than that of TPP1 whose

amount does not change through CE separation period. This

indicates that the concentration of [SSB-s31] has been

decreasing with the increasing of the migration time arising

from its on-capillary dissociation reaction. The complex and

its constituents in a sample solution begin to move as an

independent band according to the difference in their

electrophoretic mobility in the capillary at the same instant

as the beginning migration. As a result, the concentration of

free SSB or free ssDNA in the band of [SSB-s31] in the

capillary decrease greatly during the CE separation process

when the electrophoretic buffer containing no constituents

the complex was used. This separation-induced concentra-

tion-jump-like environment is the driving force of the

on-capillary dissociation reaction of the complex in CER.

[SSB-s31] and free SSB was involved in the injected sample

solution. Just after the electrophoresis is started, the zone

overlapping between [SSB-s31] and free SSB possibly retard

the complex dissociation. In fact, this likelihood is negligible

because each zone length is only 0.8 mm and the zone over

lapping time is accounted merely around 1% for the

migration time for all cases. Then the reaction time of

the on-capillary dissociation reaction can be considered to be

almost equal with the migration time. The peak height

of [SSB-s31] decreases as the migration time increase since

the degree of the decrease of [SSB-s31] through the on-

capillary dissociation reaction is proportional to the degree

of the decrease of the peak height of [SSB-s31].

As shown in Figs. 3B and C, decrease in the peak height

of the complexes with increase of migration time according

to on-capillary dissociation reaction of the complexes was

also observed in the case of both [SSB-s25] and [SSB-s20] the

same as the case of [SSB-s31]. The degree of the decrease of

the peak height of [SSB-s31], [SSB-s25] and [SSB-s20]

increased in that order. This indicates that the magnitude of

the dissociation rate constant also increase in that order.

This agrees with the results of the previous study, in which

kd of [SSB-ssDNA] decreases as the chain length of ssDNA

becomes long [16].

The dissociation reaction of [SSB-ssDNA] during CE

separation process follows the first-order kinetics. The

reaction scheme given as follows:

½SSB� ssDNA� �!kdSSBþ ssDNA ð1Þ

The basic idea of dissociation kinetic analysis by CER is to

measure the residue of the analyte complex with increasing

migration time, tm. In the kinetic analysis by CER, the peak

height signals were employed because the peak area data can

give unreliable results due to asymmetric peak profiles

arising from the on-capillary dissociation reaction. The

asymmetric-shaped [SSB-ssDNA] peak is not composed of

[SSB-ssDNA] alone, i.e. the fronting region of the peak

originates in free SSB generated by above on-capillary

dissociation reaction of Eq. 1. It is quite difficult to identify

the boundary between the region of the complex and that of

free SSB derived from the on-capillary dissociation reaction

clearly. Therefore, the dissociation degree of biomolecular

complexes is estimated not by the decrease in the peak area

of the complex but that of the peak height absorbance

of the complex as a function of reaction time for accurate

estimation. The dissociation degree-time profiles for the

on-capillary dissociation of three complexes ([SSB-s31], [SSB-

s25] and [SSB-s20]) were obtained from several CE experi-

ments with different migration time typically as shown in

Fig. 3. The double standardization method [7] is employed

for accurate measurement of the dissociation process of

[SSB-ssDNA]. The two types of standards, TPP1 and Tf,

Figure 3. Series of electropherograms of [SSB-s31], [SSB-s25], and [SSB-s20] with different migration time. A: [SSB-s31]; B: [SSB-s25];C: [SSB-s20]. Sample: 0.12 mg/ml SSB, 4.55 mg/ml ssDNA (s31, s25, and s20), 10mM phosphate buffer (pH 7.2), and 0.25 mM TPPCl. Thevalues of applied voltage are shown in each electropherogram. Other conditions were the same as for Fig. 1.



employed are both kinetically inert, i.e. their concentration

does not change through CE experiment. Typically as shown

in Fig. 3, the execution of several CE experimental runs is

necessary to obtain the series of electropherograms with

different migration times. Then TPP1 is added as an

internal standard for correcting the difference in injection

volume of each experimental run [7]. On the other hand, an

external standard, Tf, is used for an accurate estimation of

the remaining concentration of [SSB-ssDNA] that survived

during migration in the capillary, because the decrease in

the peak height of the complex arises from both the on-

capillary dissociation reaction and the zone broadening

caused by the diffusion. If the decrease in the peak height by

zone broadening is not corrected, the decrease in the peak

height by the on-capillary dissociation reaction is over-

estimated. Hence, it is necessary to correct a decrease in the

peak height by the zone broadening caused by the diffusion

of the solute. The degree of the zone broadening by diffusion

depends on the migration time and the molecular size of the

solute [7, 17]. Tf was, therefore, chosen as the external

standard, which has close migration time and molecular

weight to those of [SSB-ssDNA]. Typical electropherograms

of Tf varying with the applied voltage were shown in Fig. 4.

The electropherograms of Tf were acquired in the same CE

condition as the electropherograms of the complexes that

paired with each of them. The normalized peak height

signals of [SSB-ssDNA] and TPP1 with that of Tf in each

electropherograms are HSSB-ssDNA and HTf, respectively. In

this case, the residual ratio of [SSB-ssDNA] can be estimated

using HSSB-ssDNA and HTf,

½SSB� ssDNA�=½SSB� ssDNA�0 ¼ A HSSB�ssDNA=HTf

ð2Þ

where, [SSB-ssDNA]0 is the initial concentration of [SSB-

ssDNA] and A is the proportional constant [9]. As the

dissociation reaction follows the first-order kinetics, the rate

law is given by,

d½SSB� ssDNA�=dt ¼ kd½SSB� ssDNA� ð3Þ

Integrating Eq. 3 from t 5 0 to tm yields Eq. 4, and then

Eq. 5.

lnð½SSB� ssDNA�=½SSB� ssDNA�0Þ ¼ �kd tm ð4Þ

½SSB� ssDNA�=½SSB� ssDNA�0 ¼ expð�kd tmÞ ð5Þ

Eq. 6 is given by introducing Eq. 2 into Eq. 5.

HSSB�ssDNA=HTf ¼ a expð�kd tmÞ ð6Þ

Where, a is 1/A. Here we can obtain kd by fitting Eq. 6 using

the data HSSB-ssDNA/HTf at various tms. Thus, kd was

obtained with analyzing the dissociation degree-time profile

of [SSB-ssDNA] as the plot of the normalized peak height

signals of [SSB-ssDNA] with that of Tf (HSSB-ssDNA/HTf)

against the migration time. Simple first-order decay

profiles for [SSB-s31], [SSB-s25] and [SSB-s20] are observed

as shown in Fig. 5, and plots are fitted well to Eq. 6. The

dissociation rate constants for [SSB-s31], [SSB-s25] and

[SSB-s20] are determined to be 3.99� 10�4, 4.82� 10�4

and 1.50� 10�3/s, respectively.

The kd values found and those obtained by various

methods are summarized in Table 1 [11, 16, 18]. In

comparison with the kd values of the longer (70) [16] and

shorter (15) [11] chain length of their pair ssDNA, kd values

found were located in the middle of them. The kd values

found also decrease with increase of the chain length of

ssDNA. It is thus reasonable because kd decreases as the

chain length of it is pair ssDNA becomes long [16].

Although the values of kd of the complexes of 8, 16, and 35

bases ssDNA were also reported with stopped-flow method

and temperature-jump method [16], they do not agree with

those obtained by CER, CE and SPR as summarized in

Table 1. ‘‘Small’’ kd value of the order of 10�4/s obtained

by CER experiments show that the dissociation reaction of

[SSB-ssDNA] is a relatively slow process. This is also

supported by the results obtained by other methods [12, 16].

Contrastingly the formation reaction of the complex would

be a fast process judging from ‘‘large’’ values of the

formation reaction rate constant, kf [16, 18]. In the conven-

tional methods including stopped-flow method and

temperature-jump method, both the formation and disso-

ciation processes occur simultaneously since the experi-

ments for kinetic measurement are performed in the closed

batch system. Then, kf and kd are obtained simultaneously.

Though the simultaneous determination of kf and kd is

theoretically possible, it is difficult from a practical stand-

point to evaluate both kf and kd accurately from the experi-

mental data. The error in the evaluation of kd would be very

TfTPP+

Tf

Tf 20 kV

9 kV

5 kV

0Migration time / min

TPP+

TPP+

4 8 12 16 20

Figure 4. A series of electropherograms of Tf with differentmigration time. Sample: 0.18 mg/ml Tf, 10 mM phosphate buffer(pH 7.2), and 0.027 mM TPP1. The values of applied voltage areshown in each electropherogram. Other conditions were thesame as Fig. 1.



large since the value of kd is too small compared with that of

kf. In fact, kd of the complex with ssDNA of 35 bases was not

obtained by stopped-flow method and temperature-jump

method [16]. On the other hand, in the case of the deter-

mination of kd by the proposed method, CE [11, 12] and SPR

[18], only the dissociation process of the complex is observed

and the formation reaction does not occur since these

methods are based on separation. Abovementioned separa-

tion-induced concentration-jump-like environment, in

which the concentration of free SSB or free ssDNA around

[SSB-ssDNA] greatly decreases during separation process, is

the driving force of the dissociation reaction of the complex

in those systems, and the reverse reaction, i.e. formation

reaction, does not occur because the products of the disso-

ciation reaction of the complex (free SSB and free ssDNA)

are separated immediately. Comparing with conventional

‘‘indirect’’ measurement methods, an accurate measure-

ment of kd becomes possible by observing the dissociation

reaction of the complex directly in these separation-based

‘‘direct’’ measurement methods. The high separation ability

of CE gives the clear-cut concentration-jump-like property

suitable for direct observation of the dissociation process of

the complex to the CE-based kinetic measurement methods

but the sharp concentration-jump-like property is not

obtained in the SPR-based method [5], because diffusion

from the solid phase to the mobile phase by the concen-

tration gradient is a driving force of the separation. The

clear-cut resolution due to CE separation enables more

accurate kd measurement with CE-based kinetic analysis

methods than that of SPR method. In addition, the asym-

metric, i.e. fronting or tailingshaped, target complex peak

resulting from on-capillary dissociation reaction of the

complex is observed in CER [7] and other CE-based kinetic

analysis method similar to CER [9, 11, 12]. In the estimation

of kd from the ratio of the peak area [11] or the fitting the

asymmetrical part of electropherogram [12], the reaction

profile might not be accurately described, because the

boundary between the region of the complex and that of the

decomposition products is not clear-cut, and the region of

complex and that of the decomposition product in the peak

is not clearly distinguished. The kinetic analysis using the

peak height absorbance of the complex therefore provides

the correct description for the dissociation degree-time

profile of the complexes [7, 9], if the mutual separation

between the complex and the decomposed products is

achieved. CER is a method that can measure kd most

accurately among the existence methods for the measure-

ment of kd.

kd of the chain length of its pair ssDNA around 30 is

not estimated correctly except for our present study. SSB

binds and stabilizes template ssDNA in cooperation with

many other SSB molecules, and one SSB molecule binds

ssDNA through about 30 nucleotides [14]. SSB coming

free from template ssDNA chain binds with ssDNA again.

This cycle is repeated until the end of the DNA replication.

The half-life of [SSB-ssDNA] is calculated to be about

10 min from above kd. The rate of the replication of DNA is

reported to be about 100–1000 base pair per second [19].

The rate of the dissociation reaction of [SSB-ssDNA] is

much smaller than that of the reproduction of DNA. It is

therefore found that template ssDNA has been stabilized

by SSB as [SSB-ssDNA] during the period of the replication

of DNA.

4 Concluding remarks

In this study, the successful application of CER to the

determination of the dissociation kinetics of [SSB-ssDNA]

has been demonstrated. The dissociation rate constant of

[SSB-ssDNA] in a homogeneous solution can be measured

directly and accurately by CER without any troublesome

procedures such as immobilization and derivatization. If the

proper internal and external standard materials are avail-

Table 1. Dissociation rate constants of [SSB-ssDNA] obtained

by various methods

kd/s Chain length of ssDNA Method Reference

1.3� 10�5 70 SPR [18]

3.99� 10�4 31 CER This work



3.3� 10�2 15 CE [11]

o 1 30–40 Stopped-flow [16]

40 16 Temperature jump [16]

1700 8 Temperature jump [16]

0

0.2

0.4

0.6

0.8

1

0

Migration time / s

[SSB-s31]

[SSB-s25]

[SSB-s20]

200 400 600 800 1000

H [S

SB

-ssD

NA

] / H

Tf

Figure 5. Reaction profiles of [SSB-s31], [SSB-s25], and[SSBs20] obtained by CER. Solid lines show the approximationcurve for the first order dissociation reaction profile of eachcomplex by Eq. 6.



able, CER can actually extend its applications to a great

number of biomolecular complexes. CER can monitor

relatively slow reaction with kd in the range from 10�4 to

10�3/s. On the other hand, we had demonstrated the direct

monitoring of the dissociation reaction profile of a

polyaminocarboxylate complex with lanthanide within some

seconds to some tens of seconds and its dissociation kinetic

analysis by microchip CE reactor (mCER) [9]. If mCER is

applicable to the determination of the dissociation kinetics

of biomolecular complexes, we can determine kd of

biomolecular complexes in a wide range underlying from

10�4 to 10�1/s using CER and mCER. In CER, one must

repeat several CE runs with a variety of migration times to

obtain a set of data necessary for kinetic analysis of the

complex. But, one can acquire the data for kinetic analysis

during a single run in mCER system since UV absorption

linear imaging detection employed in mCER allows the

acquisition of a kind of continuous time-resolved electro-

pherograms [9]. CER and mCER will bring good knowledge

for shedding light on the biological functions of such

biomolecular complexes.

The authors acknowledge Dr. Akira Tsuyoshi for his usefuldiscussions and suggestions. This work was partly supported by agrant-in-aid for Scientific Research (20550070) from the JapanSociety for the Promotion of Science (JSPS).

The authors have declared no conflict of interest.

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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