Appl Biochem Biotechnol (2013) 169:268–280 DOI 10.1007/s12010-012-9972-5

Inactivation of Recombinant Human Brain-Type Creatine Kinase During Denaturation by Guanidine Hydrochloride in a Macromolecular Crowding System Yong-Qiang Fan & Hong-Jian Liu & Chang Li & Yu-Shi Luan & Jun-Mo Yang & Yu-Long Wang Received: 27 February 2012 / Accepted: 6 November 2012 / Published online: 22 November 2012 # Springer Science+Business Media New York 2012

Abstract In this study, we quantitatively examined the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000) and dextran 70, on guanidine hydrochloride (GdnHCl)-induced denaturation of recombinant human brain-type creatine kinase (rHBCK). Our results showed that both PEG 2000 and dextran 70 had a protective effect on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in the retention of 35.33 % of rHBCK activity after 4 h of inactivation, while no rHBCK activity was observed after denaturation in the absence of macromolecular crowding agents. The presence of PEG 2000 and dextran 70 at a concentration of 100 g/L could decelerate the k2 value of the slow track to 21 and 33 %, respectively, in comparison to values obtained in the absence of crowding agents. Interestingly, inactivation of rHBCK in the presence of 200 g/L PEG 2000 followed first-order monophasic kinetics, with an apparent rate constant of 8×10−5 s−1. The intrinsic fluorescence results showed that PEG 2000 was better than dextran 70 at stabilizing rHBCK conformation. In addition, the results of the phase diagram indicate that more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but the protective effects of PEG 2000 on the inactivation and unfolding of rHBCK tended to increase as the ratio of PEG 2000 increased in the mixed crowding agent solution. Though it is not clear which Y.-Q. Fan : H.-J. Liu : Y.-S. Luan School of Life Science and Biotechnology, Dalian University of Technology, 116024 Dalian, People’s Republic of China C. Li School of Life Science, Tsinghua University, 100084 Beijing, People’s Republic of China J.-M. Yang (*) Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, 135-710 Seoul, South Korea e-mail: [email protected] Y.-L. Wang (*) Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, 314006 Jiaxing, People’s Republic of China e-mail: [email protected]

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crowding agents more accurately simulated the intracellular environment, this study could lead to a better understanding of protein unfolding in the intracellular environment. Keywords Human brain-type creatine kinase . Macromolecular crowding . Guanidine . Hydrochloride denature . Inactivation . Kinetics Abbreviations CK Creatine kinase HBCK Human brain-type creatine kinase rHBCK Recombinant HBCK GdnHCl Guanidine hydrochloride PEG 2000 Polyethylene glycol 2000 Maximum emission wavelength lmax Introduction Over the past four decades, the unfolding and refolding of proteins in vitro have been extensively characterized under dilute experimental conditions with low concentrations of proteins. However, this environment differs from that encountered within living cells, where there is heavy crowding due to high concentrations of soluble and insoluble macromolecules that include proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides) [1–3]. The concentration of a single macromolecule is not so high, but overall, the macrosolute present in the cell occupies a significant part of the total volume of the medium, which is estimated to account for 30 % of the cellular volume [4]. The concentration of macromolecules in the cytoplasm is in the range of 50–400 g/L, which reduces the accessible volume in the cell [5]. Such conditions in the living cell have been defined as “macromolecular crowding” or “volume-occupied,” rather than “concentrated” [6]. It has been proposed that the effect of a crowded intracellular environment on biochemical reactions can be significant, and this is typically explained by the excluded volume effects theory [7, 8]. A variety of “macromolecule crowding agents,” including dextrans, Ficoll, polyethylene glycol, nucleic acids, and different proteins, have been used to examine the effects of volume exclusion [1, 9, 10]. The effect of macromolecular crowding on the refolding of proteins has been analyzed [11–14]; however, there have not been many biochemical studies on the effect on protein unfolding. Creatine kinase (CK; adenosine triphosphate [ATP]; creatine N-phosphotransferase, EC 2.7.3.2) reversibly catalyzes the transfer of a γ-phosphoryl from Mg2+ATP to creatine and generates adenosine diphosphate, phosphocreatine, and a hydrogen proton [15]. Therefore, CK is critically involved in energy metabolism, as well as homeostasis, and is distributed in tissues that require a lot of energy. Several types of CK have been shown to be expressed in various tissues: the muscle and brain types of CK are the most common, and three different isoenzymes including CK-MM (the muscle-type homodimer), CK-BB (the brain-type homodimer), and CK-MB (the muscle-type plus brain-type heterodimer) originate from these two common types [16, 17]. CK is an attractive model for studying protein unfolding and refolding. The unfolding of CK induced by guanidine hydrochloride (GdnHCl) has been studied extensively [18–20]; however, study of the effect of macromolecule crowding on human brain-type creatine kinase (HBCK) inactivation has not yet been conducted. In the present study, polyethylene glycol 2000 (PEG 2000) and dextran 70 were used as model crowding agents to examine the effects of macromolecular crowding on the inactivation of recombinant HBCK (rHBCK) during denaturation by GdnHCl. The equilibrium and kinetics of the inactivation of rHBCK were quantitatively studied. In addition, the

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intrinsic fluorescence spectra of partial unfolding of rHBCK were also measured. The goals of this investigation were to reinforce the growing appreciation for the need to examine the inactivation of proteins in an environment that mimics the intracellular milieu in order to understand the process as it occurs inside the cells.

Materials and Methods Materials PEG 2000, dextran 70, Ficoll 70, ATP, creatine, magnesium acetate, thymol blue, and GdnHCl were all purchased from Sigma. The other chemicals were local products of the highest analytical grade. All reagent solutions were prepared in 10 mM Tris–HCl buffer (pH 8.0). Enzymes The HBCK gene was cloned into the pET21b expression vector, expressed in Escherichia coli BL21 (Promega), and purified as previously reported by this laboratory [21]. The purified rHBCK was homologous on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme concentration was determined using the Bradford assay with bovine serum albumin (BSA) as the standard protein [22]. Unfolding of rHBCK at a Low Concentration of GdnHCl Inactivation of rHBCK was carried out by incubation of the protein dissolved in Tris–HCl (pH 8.0) with 0.5 M GdnHCl in the absence or presence of crowding agents at 25 °C. The final concentration of rHBCK was 0.2 mg/ml for most experiments, unless mentioned otherwise. Assay for rHBCK Activity The rHBCK activity was measured by the pH–colorimetry method [23]. The substrate was composed of 24 mM creatine, 4 mM ATP, 5 mM magnesium acetate, 0.01 % thymol blue, and 5 mM glycine–NaOH (pH 9.0). The reaction contained 1 ml of substrate and 10 μl enzyme solution. Proton generation was followed by monitoring the absorbance change of the indicator at 597 nm at 25 °C. All enzyme activities were normalized against and expressed as a percentage of 0.2 mg/ml solution of native rHBCK in 10 mM Tris–HCl buffer (pH 8.0). All experiments were carried out on a Helios Gamma Spectrophotometer (Thermo Spectronic, England). Determination of Kinetic Constants The time courses of rHBCK denaturation with 0.5 M GdnHCl in the presence or absence of crowding agents fit well with first-order biphasic kinetics, and the time course of inactivation was fitted to a double exponential model, using OriginLab Origin 8.5 software: A ¼ A1 ek1 t þ ð100  A1 Þek2 t . Here, A is the activity remaining; A1 is the percentage of denaturation in the fast phase; and k1 and k2 are apparent constants of the fast and slow phases, respectively. Semilogarithmic plots of the kinetic courses were used to determine apparent rate constants of the reactions fitting to the first-order monophasic kinetics. Each kinetic constant was calculated as an average of at least three repetitions.

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Intrinsic Fluorescence Assay The intrinsic fluorescence was measured on a Hitachi F-2500 fluorescence spectrophotometer with a 1-cm path length cuvette; the excitation and emission wavelengths were 280 and 300–400 nm, respectively. All the resultant spectra were collected at 25 °C in 10 mM Tris– HCl buffer (pH 8.0). The phase diagram of the fluorescence data was constructed using a previously published procedure [24]. In brief, the fluorescence intensities at 320 nm (I320) and 365 nm (I365) were normalized to the maximum of each data set, and the diagram was constructed by plotting I365 versus I320. The measurements were repeated at least three times.

Results Effects of Macromolecular Crowding Agents on the Activity and Structure of rHBCK Three crowding agents, PEG 2000, dextran 70, and Ficoll 70, were examined in this study. Three different concentrations of each macromolecular crowding agent, 50, 100, and 200 g/ L, were tested. The relative activities of rHBCK were measured after 1 h incubation at 25 °C in the absence or presence of crowding agents. The results (Fig. 1) showed that different concentrations of PEG 2000 had almost no significant effect on enzyme activity compared with the control samples that did not include macromolecular crowding agents. The results were the same for 50 and 100 g/L of dextran 70. However, 200 g/L dextran 70 and all three concentrations of Ficoll 70 significantly affected enzyme activity, with a >32 % reduction in activity after 1 h of incubation. In addition, we found that there was a significant effect of Ficoll 70 on the structure of rHBCK (data not shown). Protein crowding agents, such as BSA, and nucleic acid crowding agents, such as calf thymus DNA (CT DNA), are more relevant from a physiological point of view than are polysaccharide crowding agents; however, the concentration of BSA and CT DNA significantly disturbed the measurement of intrinsic fluorescence of rHBCK. Therefore, PEG 2000 (50, 100, and 200 g/L) and

Fig. 1 Effects of different macromolecular crowding agents on the activity of native rHBCK. ρ denotes the concentration

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dextran 70 (50 and 100 g/L) were employed as single crowding agents in the following experiments. Effects of Single Macromolecular Crowding Agents on Inactivation of rHBCK at Low Concentration of GdnHCl As shown in Fig. 2, in the absence of crowding agents, the inactivation of rHBCK in 0.5 M GdnHCl at 25 °C was very rapid in the first 10 min, with a diminution of more than 60 % of its activity, and full inactivation occurred after 4 h. The results in Fig. 2a, b indicate that both PEG 2000 and dextran 70 had protective effects on the inactivation of rHBCK induced by 0.5 M GdnHCl at 25 °C. The presence of 200 g/L PEG 2000 resulted in 46 and 35 % of activity retention after the 1- and 4-h inactivations, respectively. Moreover, the results suggest that the protective effect of PEG 2000 is concentration-dependent. Contrary to PEG 2000, dextran 70 at a concentration of 100 g/L, retained 13 % of rHBCK activity (Fig. 3a), which was slightly lower than that retained with 100 g/L PEG 2000. In the absence of crowding agents, the time course of inactivation followed first-order biphasic kinetics (Fig. 2b), with the kinetic parameters shown in Table 1. The rate constant of the fast phase, k1, was about 50-fold higher than k2 of the slow phase. In the presence of PEG 2000 or dextran 70, the time courses of inactivation with 50 and 100 g/L of either crowding agent also followed first-order biphasic kinetics (Figs. 2b and 3b, respectively). In comparison to the results in the presence of dextran 70, there was more activity remaining for the inactivation in the presence of PEG 2000 at the end of the fast phase (Table 1). Interestingly, the reaction with 200 g/L PEG 2000 followed first-order monophasic kinetics (Fig. 2b, c), with an 8×10−5 s−1 apparent rate constant (Table 1). As shown in Table 1, the presence of PEG 2000 and dextran 70 decreased the value of k2 to 21 and 33 % with 100 g/L of crowding agents, respectively. Effects of Mixed Macromolecular Crowding Agents on Inactivation of rHBCK at Low Concentration of GdnHCl To compare the inactivation of rHBCK in the presence of single crowding agents with that in mixtures of crowding agents, we made mixtures of crowding agents at 100 g/L total concentration composed of both PEG 2000 and dextran 70 at three ratios, 1:9, 1:1, and 9:1. As shown in Fig. 4, the mixed crowding agents all had a protective effect on the inactivation of rHBCK. The extent of rHBCK inactivation decreased slightly with the increasing ratio of PEG 2000 in the mixed crowding agents; however, these differences were not significant. The time courses of inactivation of mixed crowding agents are plotted in Fig. 4b, and the inactivation rate constants were listed in Table 2. Compared with 100 g/L of either crowding agent alone, the mixed crowding agents had almost the same protective effect on rHBCK inactivation, and the same conclusion was made based on the time course results. Therefore, it seems that the protective effects are correlated with the concentration of the mixed crowding agents, not the ratio of macromolecular agents. Effects of Macromolecular Crowding Agents on Intrinsic Fluorescence Spectra of GdnHCl-Induced Unfolding of rHBCK The rHBCK was denatured with 0.5 M GdnHCl for 4 h in 10 mM Tris–HCl buffer containing different concentrations of macromolecular crowding agents. The intrinsic fluorescence spectra are shown in Fig. 5, showing that, in 0.5 M GdnHCl, the red shift of

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Fig. 2 Inactivation of rHBCK in the absence and presence of PEG 2000. a Effects of different concentrations of PEG 2000 on the inactivation of rHBCK induced by 0.5 M GdnHCl for 4 h at 25 °C The data with error bars are expressed as the mean±SD (n03). b Kinetic inactivation time course of rHBCK induced by 0.5 M GdnHCl in the absence (filled squares) and presence of 50 g/L (filled inverted triangles), 100 g/L (filled triangles), or 200 g/L (filled circles) PEG 2000 within 1 h. c The semilogarithmic plot of activity versus time in the presence of 200 g/L PEG 2000. The data in c was obtained from b

rHBCK’s maximum peak was from 333 to 336.5 nm in the absence of crowding agents. Increasing PEG 2000 concentration prevented conformational changes of partially unfolded rHBCK such that there was almost no change in the peak position when using 200 g/L PEG 2000. The results also showed that the PEG 2000 concentration played an important role in the rHBCK unfolding process. Compared with PEG 2000, dextran 70 produced a weaker protective effect on the conformational

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Fig. 3 Inactivation of rHBCK in the absence and presence of dextran 70. a Effects of different concentrations of dextran 70 on the inactivation of rHBCK induced by 0.5 M GdnHCl for 4 h at 25 °C The data with error bars are expressed as the mean±SD (n03). b Kinetic inactivation time course of rHBCK induced by 0.5 M GdnHCl in the absence (filled squares) and presence of 50 g/L (filled circles) and 100 g/L (filled triangles) dextran 70 within 1 h

change of partially unfolded rHBCK (Fig. 5b). An identical trend in protective effect was also obtained when 0.7 M GdnHCl was used (Fig. 5d, e). We also examined the effects of mixed crowding agents consisting of different ratios of PEG 2000 and dextran 70 on intrinsic fluorescence spectra of unfolded rHBCK induced by 0.5 M GdnHCl (results in Fig. 5c). The concentrations and mixing ratios of the crowding agents were the same as those described in the “Effects of Mixed Macromolecular Crowding Agents on Inactivation of rHBCK at Low Concentration of GdnHCl” section. The results indicated that the Table 1 Inactivation rate constants for rHBCK in the absence and presence of single crowding agents Macromolecular crowding concentration (g/L)

A1 (%)

k1 (×10−3 s−1)

k2 (×10−3 s−1)

Absent

45±2

29.2±12.1

0.58±0.21

21±2 15±1

34.9±7.5 29.5±6.0

0.46±0.24 0.48±0.26

PEG 2000

Dextran 70

50 100 200a

–

0.08±0.01

50

26±3

30.6±5.2

0.38±0.08

100

24±4

21.5±3.5

0.39±0.06

Data are presented as the mean±SD (n03) a

Reaction was monophasic

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Fig. 4 Inactivation of rHBCK in the absence and presence of mixed crowding agents. a Effects of mixed crowding agents on inactivation of rHBCK induced by 0.5 M GdnHCl for 4 h at 25 °C Residual activity of rHBCK is expressed as a function of the ratio (in percent) of the weight of PEG 2000 to the total weight of mixed crowding agents. The data with error bars are expressed as the mean±SD (n03). b Kinetic inactivation time course of rHBCK induced by 0.5 M GdnHCl in the absence (filled squares) of crowding agent and in the presence of 0 g/L (filled triangles), 10 g/L (filled inverted triangles), 50 g/L (filled diamonds), 90 g/L (filled stars), and 100 g/L (filled circles) PEG 2000 within 1 h

optimal ratio of PEG 2000 to dextran 70 for preventing conformational changes of rHBCK unfolding was 9:1. An identical trend in protective effect was obtained when 0.7 M GdnHCl was used (Fig. 5f).

Table 2 Inactivation rate constants for rHBCK in the presence of single or mixed crowding agents Mixed crowding agent concentration (g/L)

A1 (%)

k1 (×10−3 s−1)

k2 (×10−3 s−1)

PEG 2000

Dextran 70

0

100

24±4

21.5±3.5

0.39±0.06

10

90

23±1

17.9±6.8

0.45±0.23

50

50

24±1

13.7±6.3

0.39±0.18

90

10

23±2

11.4±3.2

0.41±0.12

100

0

15±1

29.5±6.0

0.48±0.26

Data are presented as the mean±SD (n03)

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Fig. 5 The intrinsic fluorescence emission spectra of unfolded rHBCK induced by 0.5 and 0.7 M GdnHCl in the absence and presence of macromolecular crowding agents. a, d Native rHBCK was added to the unfolding buffer containing 0.5 M (a) or 0.7 M (d) GdnHCl and 50 g/L (curve 3), 100 g/L (curve 4), and 200 g/L (curve 5) PEG 2000. b, e Native rHBCK was added to the unfolding buffer containing 0.5 M (b) or 0.7 M (e) GdnHCl and 50 g/L (curve 3) and 100 g/L (curve 4) dextran 70. c, f Native rHBCK was added to the unfolding buffer containing 0.5 M (c) or 0.7 M (f) GdnHCl and 100 g/L mixed crowding agents. The intrinsic fluorescence emission spectra in the presence of mixed crowding agents containing 0 g/L (curve 3), 10 g/L (curve 4), 50 g/L (curve 5), 90 g/L (curve 6), and 100 g/L (curve 7) PEG 2000 are listed, respectively. Intrinsic fluorescence emission spectra of unfolded rHBCK were measured after 4 h (a–c) or 0.5 h (d–f). Native and partially unfolded rHBCK in the absence of crowding agents are labeled as curve 1 and curve 2, respectively. The inset plots show the maximum wavelength of rHBCK in the presence of macromolecular crowding agents of various concentrations

As 200 g/L PEG 2000 best prevented the conformational change of unfolding rHBCK, we examined the denaturation of rHBCK in buffer containing a series of concentrations of GdnHCl (0 to 3.5 M) for 12 h at 4 °C in the absence or presence of 200 g/L PEG 2000, and the results are shown in Fig. 6. The red shift of the rHBCK maximum peak was from 332.5 to 344.5 nm in the presence of PEG 2000 and from 332.5 to 349 nm in the absence of PEG 2000. To probe the differences in the stepwise changes in GdnHCl-induced rHBCK denaturation in the absence and presence of PEG 2000, we constructed phase diagrams to detect the asynchronous changes of the hydrophobic (monitored by I320) and hydrophilic fluorophores (monitored by I365), and the results are shown in Fig. 7. In the absence of PEG 2000, the diagram of rHBCK denaturation induced by GdnHCl was composed of four linear parts with joint positions at 0.5, 1.4, and 2.8 M, respectively (Fig. 7a). Figure 7b clearly shows a phase diagram where the presence of 200 g/L PEG 2000 consisted of five linear parts with joint positions at 0.7, 1.2, 2.4, and 3.0 M, respectively. These results indicate that GdnHClinduced unfolding of rHBCK in the presence of PEG 2000 is an exceptionally more complex process compared with that in the absence of crowding agents.

Discussion CK is an important enzyme in cellular energy metabolism [25]. The unfolding of CK has been studied extensively in vitro under dilute experimental conditions; however, the macromolecular crowding agents reflecting the physical environment under which the unfolding

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Fig. 6 The changes in the maximum emission wavelength of unfolding rHBCK induced by different concentrations of GdnHCl in the absence (filled circles) and presence (filled squares) of 200 g/L PEG 2000

of CK occurs is still of great interest to researchers. Usually, the agents used for mimicking macromolecular crowding should be inert with respect to the target proteins. BSA, CT DNA, Ficoll 70, PEG 2000, and dextran 70 had been used as crowding agents in studies of the refolding of CK-MM [1, 9, 26]. The work of Lin [9] found that PEG 2000 had a significant effect on the activity of native CK-MM, with Ficoll 70 and dextran 70 also performing well. In contrast, the results of the present work show that Ficoll 70 and a high concentration of dextran 70 have a significant effect on the activity of native rHBCK, while PEG 2000 has almost no effect. These results indicate that there are some differences in the characteristics of different types of CK, and studying these differences in a crowding system will help us understand the proteins under more realistic conditions. In addition, it is better to examine the crowding agents carefully to make sure they are inert with respect to the target protein. According to Minton’s crowding theory [27], macromolecules exclude the available cell volume and reduce conformational entropy, resulting in increases in the free energy of a solution and chemical potential of all molecules present in that solution. The main result is to favor the formation of a state that excludes the least amount of volume for all other

Fig. 7 Phase diagram of fluorescence representing the unfolding of rHBCK induced by increasing GdnHCl concentration in the absence (a) and presence (b) of 200 g/L PEG 2000. Denaturant concentration values in molars are indicated in the vicinity of the corresponding symbol. Each straight line represents an all-or-none transition between two conformers of rHBCK. The inactivation conditions are the same as those for Fig. 6

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macromolecules present. The biochemical data on the unfolding of rHBCK in the present study under crowding conditions are consistent with the excluded volume theory. The results (Figs. 2a and 3a) of equilibrium inactivation of rHBCK showed that the macromolecular crowding agents, PEG 2000 and dextran 70, decreased the extent of rHBCK inactivation in the presence of GdnHCl, and the protective effect increased as the macromolecular crowding agent concentration increased. The stabilizing effect of crowding on activity is largely restricted to macromolecules [28], and the effects of crowding on reaction rate are complex and depend crucially on the precise nature of the reaction and on the degree of crowding [3]. The quantitative results (Table 1) suggest that PEG 2000 and dextran 70 could decelerate the slow track of rHBCK inactivation to 61 and 52 %, respectively, at concentrations of 100 g/L compared with values observed in the absence of crowding agents. We conclude (Figs. 2b and 3b; Table 1) that dextran 70 seemed to decrease the rate constants more dramatically in comparison to PEG 2000 at an equivalent concentration, though more residual activities were obtained when PEG 2000 was used. However, the inactivation kinetics changed from biphasic to monophasic when 200 g/L PEG 2000 was used. Such results indicate that there are different mechanisms of rHBCK inactivation in the presence of PEG 2000 and dextran 70. When we examined the GdnHCl-induced unfolding of rHBCK in the macromolecular crowding system, we found that the conformational change was inhibited to a greater extent in the presence of PEG 2000 compared with that in the presence of dextran 70 (Fig. 5). The protective effect of 200 g/L PEG 2000 could be further demonstrated by the data from Fig. 6. The denatured rHBCK maximum peak of intrinsic fluorescence was almost consistent with that of native rHBCK when 200 g/L PEG 2000 was present. These results indicate that the protective effects of PEG 2000 on the inactivation of rHBCK are mainly based on protecting the conformation of rHBCK. The present study also indicated (Fig. 7) that the unfolding of rHBCK induced by GdnHCl in the presence of a macromolecular crowding system may be more complicated than that under dilute experimental conditions, and more intermediates should be studied. The effects of crowding agents on the unfolding of GdnHCl-denatured rHBCK are diverse and depend on the nature of the crowding agents used. There are two important aspects of the nature of crowding agents: the size and shape and the viscosity. PEG 2000 can be modeled as an effectively spherical particle, while dextran 70 is better modeled as a rod-like particle. Although the relative size of PEG 2000 is much smaller than that of dextran 70, its excluded volume effect on protein unfolding is more effective, as demonstrated here. At the same concentration, the viscosity of dextran 70 is higher than that of PEG 2000, so the factor of viscosity must be considered in order to more accurately simulate the intracellular environment. Here, we compared the effects of PEG 2000 and dextran 70 on the inactivation of rHBCK. It has been proposed [1] that a mixture of crowding agents reflects the physiological environment more accurately than individual crowding agents, so we examined the effects of using a combination of PEG 2000 (100 g/L) and dextran 70 (100 g/L). Unexpectedly, the mixed crowding agents did not produce more residual activity than the single crowding agents. Furthermore, the two agents seemed to interfere with each other when the PEG 2000/ dextran 70 ratio was 1:9, resulting in a lower residual activity than that observed for 100 g/L dextran 70. As the data after 4 h (Fig. 4a) did not show a statistical difference in the residual activity, the discrepancy may be due to the regression method used to determine the apparent rate constants or the model’s inherent assumptions. However, we found that the protective effects on the inactivation and unfolding of rHBCK increased as the ratio of PEG 2000 in the mixed crowding solution increased. In addition, the protective effects of PEG 2000 and dextran 70 seemed to be additive.

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In conclusion, we have investigated the effects of single and mixed crowding agents on the inactivation of rHBCK induced by GdnHCl by analyzing residual activity, reaction kinetics, intrinsic fluorescence, and phase diagrams. Both PEG 2000 and dextran 70 produced an increase in the residual activity and a decrease in the inactivation rate. The intrinsic fluorescence results showed that PEG 2000 could better stabilize the rHBCK conformation compared to dextran 70. In addition, more intermediates may be captured when rHBCK is denatured in a macromolecular crowding system. Mixed crowding agents did not produce better results than single crowding agents, but there seemed to be an additive effect with crowding agent mixtures. Although it has not yet established which crowding agents most accurately simulate the intracellular environment, information from this study can enhance our understanding of how rHBCK unfolds in an intracellular environment. Acknowledgments Dr. Yu-Long Wang was supported by funds from the Science and Technology Bureau of Jiaxing, Zhejiang (grant nos. 2009AZ1027 and 2011AZ1027). Dr. Jun-Mo Yang was supported by a grant of the Korea Health 21 R&D Project (Ministry of Health, Welfare and Family Affairs, Republic of Korea, 01PJ3-PG6-01GN12-0001) and a grant from Samsung Biomedical Research Institute (GL1-B2-181-1).

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