Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 206-211

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 206-211

Evidence for the Existence of Cross-Linked Intermediates during Unfolding and Refolding of CK in UGGE

WANG Xicheng , XIE Cheng ,YANG Jian  ZHOU Haimeng *

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
* To whom correspondence should be addressed, E-mail address: zhm-dbs@mail.tsinghua.edu.cn

Received: 2000-05-18

Code Number: ts01067

Abstract:   

Urea gradient gel electrophoresis (UGGE) is an important technique for studying the conformation changes of proteins during denaturation. This paper reports on an investigation of the unfolding and refolding of creatine kinase (CK) by UGGE. The native and denatured CK underwent electrophoresis in polyacrylamide gels containing a linear 0-8 mol/L gradient of urea perpendicular to the direction of migration. The results showed that unfolding and refolding of CK is a relatively rapid process. The denatured enzyme could refold to a conformation with activity during electrophoresis at low urea concentrations, indicating that denaturation in urea is reversible. More importantly, both the native and denatured CK were separated into multiple parallel bands through UGGE, but the bands decreased significantly when mercaptoethanol was added to the samples. The results suggest that various kinds of unfolding and refolding intermediates were formed during UGGE, which are assumed to be oligomers with disulfide bonds between peptide chains. Urea/SDS (sodium dodecylsulphate) polyacrylamide two-dimensional electrophoresis proved that these unfolding and refolding intermediates formed during UGGE were oligomers which were composed of different number of subunits cross-linked by disulfide bonds. The results indicate that the unfolding and refolding of CK are relatively rapid processes with some cross-linked intermediates with disulfide bonds during unfolding and refolding of the enzyme.

Key  words:  creatine kinase; unfolding and refolding; urea gradient gel electrophoresis; intermediate; disulfide bonds

Introduction   

Creatine kinase (CK) plays an important role in cell energy metabolism[1]. It catalyses the reversible transfer of a phosphoryl group from ATP to creatine generating phosphocreatine and ADP. Owing to its importance in bioenergetics, CK has been extensively studied. There are four isoforms of the enzyme: muscle type (MM), brain type (BB), hybrid type (MB), and mitochondria type (MiMi).

Muscle-CK is a dimer composed of two identical subunits, both with relative molecular mass of 4.3x10⁴. Four SH groups are located on Cys-73, Cys-145, Cys-253, and Cys-282 in each peptide chain. The SH group of Cys-282 is essential for the activity of CK[2]. There are no interchain disulfide bonds in the enzyme and it is classified into oxidized and reduced forms according to the existence of intrachain disulfide bonds. Several SH groups are oxidized to form a disulfide bond in each peptide chain of oxidized CK, which constitutes 15% of native rabbit MM-CK[3].

Urea gradient gel electrophoresis (UGGE) was developed in the 1970's. During electrophoresis, the sample migrates in a polyacrylamide slab gel with a urea gradient perpendicular to the electrophoresis direction. Therefore, protein molecules at various positions along the gel migrate in different urea concentrations. Different molecular sizes and surface charges cause the denatured protein intermediates to be separated in the gel to give a two-dimensional pattern of the conformation change[4]. UGGE can directly and qualitatively reflect the continuous process of protein denaturation. It is indispensable for studying the urea-induced unfolding and refolding of proteins.

In this paper, UGGE was used to investigate the conformational change of rabbit muscle CK in a continuous urea gradient of 0-8 mol/L. Cross-linked intermediates with disulfide bonds were found during the unfolding and refolding of urea-denatured CK.

1 Materials and Methods  

1.1 Materials

The muscle CK from rabbit muscle was prepared as described by Yao et al.[5] The specific activity is 320 mmol/(minDK·mg). Acrylamide, ammonium persulfate,  and  N,N,N'N'-tetramethyl  ethylenediamine were purchased from Bio-Rad. N'N'-methylene-bis-acrylamide and SDS were BDH products. b-Mercaptoethanol, glycine, ADP, creatine phosphate, and urea were all from Ameresco and MTT was from Biomol. ATP was obtained from Boehringer. The other reagents were local products of analytical grade without purification.

1.2 Methods

The enzyme concentration was measured using the absorption coefficient A^%₁ cm =8.8[6]. The CK activity was determined by the pH change indicator method[5] at  25 °C . A Perkin Elmer UV-VIS spectrophotometer was used to measure the absorption and enzymatic activity.

UGGE of CK was carried out as described by Goldenburg and Creighton[7]. The 0-8 mol/L urea gradient gel was made by mixing two acrylamide solutions with constant urea concentrations (0 mol/L and 8 mol/L). Since different urea concentrations cause a variable viscosity across the gel, a compensating inverse gradient of polyacrylamide concentration was included in the slab. The first solution contained 7.5% acrylamide and no urea, while the second solution included 5.5% acrylamide and 8 mol/L urea. The gel slabs were square (16 cmx16 cmx 1 mm ). After polymerization, the slab was rotated by 90°. An 1.5 cm thick layer of stacking gel solution was polymerized on top of the gel. The result was a slab gel with a urea gradient perpendicular to the electrophoresis direction. 300 mg of CK in 100 mL buffer (0.05 mol/L Tris-HCl, pH 8.0) was laid across the top of the stacking gel. The sample was denatured with 8 mol/L urea for 2 hours before loading during UGGE of denatured CK. The electrophoresis was carried out on a Bio-Rad Protein II apparatus with a constant current of 25 mA at 25 °C for 7 hours.

The urea/SDS polyacrylamide two-dimensional electrophoresis was carried out on a Bio-Rad Mini-Protein II apparatus. The first direction was 7.5% PAGE containing 3 mol/L urea  (7 cmx 8 cmx0.75 mm slab). The second direction used  12.5%  SDS-PAGE or 5%-15% gradient SDS-PAGE (7 cmx8 cmx1 mm slab). 200 mg CK in 80 mL buffer (0.05 mol/L Tris-HCl, pH 8.0) was denatured with 8 mol/L urea for 2 hours and laid on the gel. After 2 hours of electrophoresis in the first direction, a strip of gel parallel to the migration direction was cut out and treated with SDS sample buffer with mercaptoethanol (reduced, R) or without mercaptoethanol (non-reduced, NR) for 30 minutes. Then the strip was put on top of the second direction gel to begin  12.5%  or 5%-15% gradient SDS-PAGE. For the R SDS-PAGE (strip was treated with mercaptoethanol),  0.1%  mercaptoethanol was added to the electrode buffer during electrophoresis of the second direction. 200 mg native CK was also used for the urea/NR SDS polyacrylamide two-dimensional electrophoresis (strip not treated with mercaptoethanol).

Staining for CK activity was carried out as described by Font et al.[8]  The CK activity was detected using an overlay reaction gel containing  10 mg/mL  agar and 0.3 mol/L MTT. Before use, the gel was incubated with a visualization mixture containing a 100 mmol/L Tris-HCl buffer (pH  7.0 ), 20 mmol/L glucose, 10 mmol/L Mg(AC)₂, 1 mmol/L ADP, 35 mmol/L creatine phosphate,  0.6  mmol/L NADP⁺,  0.25 mg/mL PMS, 2.8 IU/mL hexokinase, and 1.4 IU/mL glucose-6-phosphate dehydrogenase. The urea-gradient gel was washed using a 100 mmol/L Tris-HCl buffer (pH 7.0) after UGGE and incubated with the reaction gel at 37 °C in the dark for 15 minutes. The reaction gel was fixed in 70% ethanol, 5% HAc and 1% glycerin.

2 Results  2.1 UGGE of creatine kinase

Figure 1(a)  shows the UGGE pattern of 0-8 mol/L urea for native CK. As the urea concentration increased from 0 mol/L to 2 mol/L, only one band was found and its mobility increased. At 2 mol/L urea, new multiple parallel bands with lower mobility appeared, while the initial band gradually disappeared at 3 mol/L and a spur of high mobility pointing towards the region of high urea concentration was seen. The mobility of these multiple bands decreased as the urea concentration increased. An abnormal increase of mobility appeared at 6 mol/L. Figure 1(b) shows the result from staining for CK activity. The band on the corresponding position in the agar gel shows that only the band with higher mobility in the range of 0-3 mol/L urea concentration had enzymatic activity.

Figure 2 (a) shows the UGGE pattern for CK denatured with 8 mol/L urea for 2 hours before loading. There are multiple parallel bands over the whole urea concentration range, which exhibit similar patterns as the native CK except with more bands. The CK activity staining is shown in Fig. 2 (b). An active band in the range of 0-3 mol/L urea indicates that the band of higher mobility at low urea concentration has enzymatic activity.

The  UGGE pattern for native CK in the presence of 2% mercaptoethanol is shown in Fig. 3. Its electrophoresis behavior was similar to that of the native enzyme at low urea concentration, but with enhanced resistance to urea denaturation. The most remarkable difference was that no multiple bands were found at high urea concentrations. The activity staining indicates the band at low urea concentration had CK activity.

Fig. 4

2.2 Urea/SDS polyacrylamide two-dimensional electrophoresis of CK

The denatured CK was separated into multiple parallel bands through PAGE containing 3 mol/L urea. When the strips cut out from the first gel were treated with reduced and non-reduced SDS sample buffers, the SDS-PAGE in the second direction showed different results. Figure 5 for the R SDS-PAGE shows that the components separated by the first direction migrated at the same speed. By comparing with the relative rate of mobility of standard proteins, their relative molecular mass 4.3 x 10⁴ for the components with lower mobility in the first direction also migrated slower in the electrophoresis in the second direction. The relative molecular mass was 4.3x10⁴ for the components with the highest mobility and 8.0x10⁴-9.0x10⁴ for the components with lower mobility. In addition, some components with larger molecular masses were seen at the interfaces between the separating and stacking gels. The result for the gradient NR SDS-PAGE, Fig.7, shows that the relative molecular masses of the components with different mobilities in the first direction were 4.3x10⁴, 8.0x10⁴-9.0x10⁴, 1.3x10⁵ and higher.

The native CK also separated into multiple parallel bands through PAGE containing 3 mol/L urea, though it had fewer bands than the denatured CK. When the strip cut out from the first gel underwent NR SDS-PAGE in the second direction, the result shown in Fig. 8 was similar to that in Fig. 6. The components with lower mobility in the first direction also migrated slower in the second direction. Their relative molecular masses were  4.3 x10⁴, 8.0x10⁴-9.0x10⁴ and higher.

3 Discussion  

The UGGE patterns show that the sample bands formed during electrophoresis were continuous which indicates that the halftime for transition of intermediate conformations is less than 1% of the separation time[4]. Thus unfolding and refolding of CK are relatively rapid processes.

The UGGE patterns of native CK show that the dimeric enzyme gradually dissociated into subunits at 0-3 mol/L urea, as evidenced by the increased mobility. At 2 mol/L urea, molecules with lower mobility appeared. Increasing the urea concentration enhanced the extent of denaturation of these molecules which caused their structure to become looser so that they migrated slower. The spur of high mobility pointing towards the region of high urea concentration shows that some CK molecules were still in their folded dimeric and monomeric forms while others had undergone an unfolding transition between 2 mol/L and 3 mol/L urea. At 6 mol/L urea, however, the mobility increased abnormally, perhaps due to the formation of intermediates with compact structure[9] or due to the exposure of inner charges.

After the UGGE of denatured CK, the band at low urea concentration had enzymatic activity, indicating that denatured CK can refold to an almost native conformation with activity through dilution in the electrophoresis, which is consistent with the recovery of activity through dilution in solution[10].

The most important phenomena revealed in these experiments are that neither the native nor the denatured CK formed multiple parallel bands during UGGE. It has been reported that UGGE of human apoA-I[11]  and a storage protein from Vitis vinifera seeds[12]  also show similar results. These individual bands rather than a smearing resulting from a variety of molecules indicate several components with different constant sizes or surface charges. The results suggest that various kinds of unfolding and refolding intermediates were formed during UGGE.

Far fewer bands appeared during UGGE when mercaptoethanol was added to the native and denatured CK. This result implies that the formation of these unfolding and refolding intermediates is related to the disulfide bonds. The enzyme structure without interchain disulfide bonds is assumed to have become looser as it migrated during denaturation and the change of the interactions between molecules resulted in a local oxidizing environment, so that the SH groups on the enzyme were oxidized to disulfide bonds between peptide chains. The different extents of disulfide bond cross-linking may result in the formation of different cross-linked oligomers (dimer, trimer, tetramer, etc.). However, the SH groups on the enzyme were protected by the reducing agent when mercaptoethanol was added during electrophoresis, so the cross-linked intermediates could no longer be seen.

Urea/SDS polyacrylamide two-dimensional electrophoresis was used to determine whether these bands were molecules with different sizes formed by interchain cross-linking.

During urea/SDS polyacrylamide two-dimensional electrophoresis, the denatured CK was separated into intermediates with different mobility in the first direction. In the R SDS-PAGE of the second direction, the disulfide bonds between the chains of cross-linked intermediates were broken to form subunit-SDS compounds. Thus, all the molecules separated in the second direction were the CK monomers with relative molecular masses of 4.3x10⁴. However, in the NR SDS-PAGE of the second direction, molecules appeared with different mobilities because the peptide chains of the intermediates were still associated by disulfide bonds. So CK monomers were obtained in the second direction together with cross-linked oligomers with relative molecular masses of 8.6x10⁴, 1.29x10⁵ and 1.72x10⁵. The result of the urea/NR SDS polyacrylamide two-dimensional electrophoresis of native CK was similar to that of denatured CK which indicates that the intermediates formed during unfolding of the enzyme were also the cross-linked oligomers. All these results confirm that the multiple bands formed during UGGE are intermediates composed of different numbers of subunits cross-linked by disulfide bonds.

The activity staining shows that only the molecules with the highest mobility at 0-3 mol/L urea concentrations possessed CK activity. Thus,  neither the monomers with loose structures nor the cross-linked oligomers have enzymatic activity.

The results can be summarized in the following conclusions:

(1) The unfolding and refolding of CK in urea are relatively rapid processes.
(2) The denaturation of CK in urea is reversible. The denatured CK can refold to an almost native conformation with activity through dilution in electrophoresis.
(3) Intermediates with different mobilities formed during both unfolding and refolding of CK in UGGE are oligomers composed of different numbers of subunits cross-linked by disulfide bonds.

References

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