MTT assay

For cytoviability, the MTT assay has been widely used as a colorimetric assay for measuring the activity of mitochondrial dehydrogenase that reduce MTT, a tetrazolium dye: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, substrate to form formazan, which is generated only by living cells but not dead cells.

In this assay, AGS cells were inoculated in 200 uL of complete growth medium at a concentration of 2 x 10⁴ cells in a 96-well microtiter plates. Plates were incubated for 24 h at 37^oC in an atmosphere of 5% CO2 in air. Different concentrations of G.h-rTDH were added and treated for 1 h before the supernatant was removed via a centrifugation separation. Fresh MTT solution (5 mg/mL) was dissolved in PBS and filtered by a 0.22 filter, and was diluted 10-fold in a complete medium. A 100 ul aliquot of the above solution was added to each well. Plates were wrapped by the aluminum foil and incubated for a further 5 h at 37 ^oC. MTT was then removed from the wells, and the formazan crystals were dissolved in 200 ul of Dimethyl sulfoxide

21

experiments.

3.13 Thioflavin T florescence assay

Thioflavin T (ThT) is a benzothiazole compound that possesses the light-emitting component of leuciferin. It can be utilized to visualize the amyloid beta content of protein in the solution. ThT binds to amyloid fibrils and the florescence intensity can identify the fibril content by florescence spectra. A 10 g sample of G.h-rTDH protein was respectively and sequentially heated to 37^oC, 70^oC and 90^oC for 15 min and then subjected to rapid cooling in ice water for 15 min.

Before measuring the florescence of the fibril content in the solution, ThT and Tris-HCl buffer were premixed. The ThT/Tris solution was as described: ThT powder was dissolved in distilled water and the final concentration was 1 mg/mL (3.14 mM).

Then, 1.6

l of ThT solution was added to 1 mL 50mM Tris-HCl, pH7.0 buffer.

Before measurement, an aliquot of 200

l ThT/Tris solution was mixed with

thermally pre-treated protein, the fiber forming protein containing beta sheets became immediately bound to ThT. The florescence of Thioflain T was measured at 460-600 nm when excited at 450 nm using fluorescence spectrophotometry (Hitachi, F-7000).

The Thioflavin T was obtained from Sigma (St. Louis, MO).

Chapter 4 Results

4.1 Cloning, sequence analysis and identification of the G. hollisae tdh gene

The nucleic acid sequences of tdh gene from various Vibrio species were aligned and analyzed to find the highly conserved sequences. The primers for the conserved gene were thus designed, and used in the polymerase chain reaction (PCR) to amplify the putative tdh gene from G. hollisae BCRC 15890 genomic DNA. The construct plasmid carry out tdh-mutated gene was obtained from the lab. The PCR amplified G.

hollisae tdh gene is 570-bp in size and encodes a polypeptide of 189 amino acids involving a signal peptide with 24 amino acids in the N-terminal region, and a mature protein of 165 residues, with a predicted molecular mass of 18,616.9 Da. Notably, three distinct amino acid changes, i.e. Tyr53His53, Thr59Ile59, and Ser63Thr63, were observed from the sequence alignment between the PCR amplified tdh gene from G. hollisae BCRC 15890 genomic DNA (assigned as G.h-rTDH^WT) and the historically published tdh gene from G. hollisae 9041 in the NCBI data bank (assigned as G.h-rTDHY53H/T59I/S63T

). Amino acids sequence alignment between the two tdh genes is shown in Figure 5.

23

FIGURE 5. The sequence alignment of putative tdh genes between the PCR amplicon tdh gene from G. hollisae BCRC 15890 genomic DNA (assigned as G.h-rTDH^WT) and the historically published tdh gene, from G. hollisae 9041 as described in the NCBI data bank (assigned as G.h-rTDHY53H/T59I/S63T

). The sequence of the signal peptide with 24 amino acids in the N-terminal is shown in red. The distinct amino acid change between G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

are red boxed.

4.2 Expression, purification, determination and hemolytic activity of G..h-rTDH

^WT

The wild type G. hollisae tdh gene G.h-rTDH^WT was cloned into the plasmid pCR^®2.1-TOPO^® and subsequently transformed into Escherichia coli BL21(DE3)(pLysS) cells for protein expression. Following the incubation for 16 h at 37^oC, the harvested cells were sonicated for the expressed protein purification using a Phenyl-Sepharose 6 Fast Flow column. After the first round of chromatographic purification for the separation of impurities from crude extraction, the homogenous protein with a molecular mass of approximately 22 kDa, as resolved by 15%

SDS-PAGE was collected from the subsequent purification on the same Phenyl-Sepharose 6 Fast Flow column (Figure 6). A single band at approximately 90

kDa was observed by 10% native-PAGE, and the hemolytic activity of this protein band suggested that it is an active tetrameric protein under physiological conditions (Figure 6). MALDI-TOF MS spectrum of peptide mapping via a trypsin digestion further confirmed the identity of G. hollisae TDH.

FIGURE 6. Purification and characterization of the G.h-rTDH^WT protein from G.

hollisae. (A) The crude protein without tdh gene insertion in a pCR^®2.1^®-TOPO plasmid was obtained in the BL21(DE3)pLysS strain (lane 1). The crude protein containing the expressed G.h-rTDH^WT in the BL21(DE3)pLysS strain was also included (lane 2). The homogenous protein with a molecular mass of ~22 kDa was obtained via two Phenyl Sepharose 6 Fast Flow chromatography runs (lane 3 and lane 4). (B) Native-PAGE of purified G.h-rTDH^WT, with a molecular mass of ~90 kDa. (C) Hemolytic activity of G.h-rTDH^WT.

25

4.3 Identification of G.h-rTDH

^WT

by MALDI-TOF-TOF MS spectrometry

The purified protein was then subjected to a MALDI-TOF MS spectrometry for the internal amino acid determination to confirm the identity of purified protein. The SDS-PAGE band corresponding to G.h-rTDH^WT was first applied to an in-gel trypsin digestion as described by Rosenfeld et al.³⁵. The cutting sites of trypsin are lysine (Lys, K) and arginine (Arg, R), respectively. After digestion, the resulting peptide mixtures were analyzed by MALDI-TOF MS. Among these peptide fragments shown in Figure 7, via a database search for the mass spectrum of the peptide and its fragment ions, the tandem mass spectrum of mono-charged precursor was observed at m/z 1024.543, 1365.788, 1690.949, 2346.302, 2953.530. Following the analysis of the highest signal by MS/MS, the sequence of this fragmental was determined to be

35VSDFWTNR⁴² of G. hollisae TDH.

FIGURE 7. (A) MALDI-TOF-TOF MS spectrum and peptide mapping of G.h-rTDH protein. (B) Tandem mass spectrum of a signal charged tryptic peptide at m/z 1024.543, was deduced from the mass differences in the y-fragment ion series and partial b-ion and a-ion.

27

4.4 Expression, purification, and hemolytic activity determination of mutated G.h-rTDH

Y53A/T59B/S63C

In order to understand the role of these distinct amino acid changes between the tdh gene from the G. hollisae BCRC 15890 strain, and the historically published tdh gene from NCBI data bank from G. hollisae 9041, seven G.h-rTDH mutants specific for different combinations of Tyr53, Thr59, or Ser 63 positions were constructed and purified. The procedures for expression, purification, identification, and hemolytic activity assays of these G.h-rTDHY53A/T59B/S63C

mutants were identical with that of Gh-rTDH^WT. These seven G.h-rTDHY53A/T59B/S63C

mutants include G.h-rTDH^Y53H, G.h-rTDH^T59I, G.h-rTDH^S63T, G.h-rTDH^Y53H/T59I, G.h-rTDH^Y53H/S63T, G.h-rTDH ^T59I/S63T, and G.h-rTDHY53H/T59I/S63T

. As shown in the 15% SDS-PAGE data, all mutated G.h-rTDHY53A/T59B/S63C

exhibited a homology protein of approximately 22 KDa. The native-PAGE coupled with the blood agar plate assay further indicated that the corresponding protein bands with molecular mass of ~90KDa possess the hemolytic activity among these G.h-rTDHY53A/T59B/S63C

mutants.

FIGURE 8. (A) SDS-PAGE of purified G.h-rTDH with a molecular mass of ~22 kDa from various G.h-rTDH mutants. (B) Native PAGE of G.h-rTDH mutants, with a molecular mass of ~90 kDa. (C) Hemolytic activity determination of purified G.h-rTDH mutants on a blood agar plate.

4.5 The temperature-dependent hemolytic activity analysis and thermostability studies of G.h-rTDH

^WT

and G.h-rTDH

Y53H/T59I/S63T

To investigate the thermostability and optimal temperature for hemolytic activity of purified G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

, a suspension of 5% human erythrocytes was incubated with G.h-rTDH protein for 1 h at different temperatures ranging from 4.0-100^oC (Figure 9). Interestingly, the hemolytic activity of G.h-rTDH^WT on human erythrocytes exhibited a common Arrhenius phenomenon,

29

additional heating over 85^oC coupled with a rapid cooling treatment. Under incubation below 55 ^oC for 30 min, the G.h-rTDH^WT protein still retained over 80% of its full activity (Figure 9 (A)). In comparison, the G.h-rTDHY53H/T59I/S63T

lost the entire hemolytic activity via a heating above 60^oC, and no recovering activity was observed after a rapid cooling treatment (Figure 9 (B)).

FIGURE 9. Thermostability assay of G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

mutants.

The relative hemolytic activities were measured at 37^oC under various temperature treatments. According to the results, G.h-rTDH^WT has the Arrhenius effect, whereas the G.h-rTDHY53H/T59I/S63T

did not. Data are presented as the means for triplicate experiments. Error bars represent the standard deviations (SD)

4.6 Comparison of hemolytic activity for G.h-rTDH

Y53A/T59B/S63C

In order to investigate the biophysical characterization of various combination mutants on Tyr53, Thr59, or Ser63 positions of G.h-rTDH, the hemolytic activity of various mutants on human erythrocytes were further studied. In Figure 10, the hemolytic activities of various G.h-rTDHY53A/T59B/S63C

mutants were examined on a 96 well plate. Decreased hemolytic activities were displayed from G.h-rTDH^Y53H/T59I, G.h-rTDH^T59I/S63T, and G.h-rTDHY53H/T59I/S63T

mutants. Moreover, the G.h-rTDH^Y53H/S63T mutant showed more dominant hemolytic activity than that observed for G.h-rTDH^WT. The hemolysis ability of G.h-rTDH^S63T and G.h-rTDH^T59I mutants were very similar, and also more dominant than that observed for G.h-rTDH^WT. The concentration of various G.h-rTDHY53A/T59B/S63C

higher 25

g/mL

caused 100% hemolysis, while lower than 0.39

g/mL, their hemolytic activity was

below the detectable level and had no effect on human erythrocytes.

FIGURE 10. Compare hemolytic activity for various G.h-rTDHY53A/T59B/S63C

mutant.

31

4.7 Comparison of the hemolytic activity of G.h-rTDH

Y53A/T59B/S63C

mutants at 37

^o

C, 70

^o

C, 90

^o

C

As illustrated above, the G.h-rTDH^WT lost its hemolytic activity under 60-80 ^oC but recovered its function via continuing to heat at 90^oC coupled with a rapid cooling.

This paradoxical phenomenon is referred to as “Arrhenius effect”. G.h-rTDH^WT was first treated with different temperature heating conditions at 37^oC, 70^oC, or 90^oC, coupled by a rapid cooling treatment, respectively. The protein structure or its biophysical characteristics were changed via these three various pretreatments with different significance. The protein structure was changed from a native form into a fiber form, then transformed to the unfold state, and recovered it to its native form via a induced cooling treatment. This conformational change may affect the biophysical properties. Some mutants, G.h-rTDH^Y53H, G.h-rTDH^T59I, G.h-rTDH^S63T, G.h-rTDH^Y53H/S63T, also exhibited this unusual phenomena, but the G.h-rTDH^Y53H/T59I, and G.h-rTDH^T59I/S63T, as well as G.h-rTDHY53H/T59I/S63T

lost this Arrhenius effect, even with the concentration elevated to 10 g/mL (Figure 11).

FIGURE 11. The hemolytic activity of various G.h-rTDHY53A/T59B/S63C

mutants after different temperature pre-treatments, coupled with a rapid cooling treatment. Data are presented as the means for triplicate experiments. Error bars represent standard deviations (SD).

4.8 Analysis of G.h-rTDH

Y53A/T59B/S63C

thermostability by difference scanning calorimetry

DSC measurements involve the heating of a sealed sample of protein solution at a constant rate of temperature increase. As long as no other process occurs in the solution that releases or takes up heat, the temperature of the solution will rise monotonically with the instrumental heating (electrical power input). The DSC profile provides much valuable information, including the temperature at which the maximum excess heat capacity occurs, called “Tmax”; the area under the curve, obtained by integration using the software supplied with the instrument, gives “the enthalpy of denaturation”; the width of the peak at half of the maximum excess heat

33

co-operativity of the protein structure³⁶. The calorimetric scan of TDH^WT andother TDH mutationsin 10 mM Kpi (pH 7.0) were characterized by a single peak. The transition temperature of those proteins from low to high were 51.8^oC (G.h-rTDH^T59I), 52^oC (G.h-rTDH^Y53H), 55.3^oC (G.h-rTDH^S63T), 56.3^oC (G.h-rTDH^Y53H/S63T), 56.6^oC (G.h-rTDH^WT), 57.1^oC (G.h-rTDH^T59I/S63T), 58^oC (G.h-rTDH^Y53H/T59I), 58.4^oC (G.h-rTDHY53H/T59I/S63T

), respectively. Those proteins without Arrhenius effect including G.h-rTDHY53H/T59I/S63T

, G.h-rTDH^Y53H/T59I and G.h-rTDH^T59I/S63T have the top three Tm values, and obtained a wider FWHM, indicating that during the unfolding process by increasing temperature the more entropy is needed to disrupt the intramolecular interaction with those proteins, and more stable and compacted structures than others were conserved in these mutants. The G.h-rTDH^T59I and G.h-rTDH^Y53H have the lowest Tm values, indicating that these proteins were more unstable than others during the heat-induced denaturation.

FIGURE 12. The DSC result. Corrected DSC thermograms of G.h-rTDHY53A/T59B/S63C

in 10 mM Kpi buffer at pH 7.0.

4.9 Analyze the secondary structure change of various G.h-rTDH

Y53A/T59B/S63C

mutants by circular dichroism spectroscopy

The change of secondary/tertiary structure caused by the thermal denaturation of G.h-rTDH in Kpi buffer (pH 7.0) was examined by circular dichroism (CD) spectra.

At temperatures below 50°C, all proteins exist in a native state characterized by a

-rich secondary structure with a pronounced minimum at 218 nm (Figure 13 (E)).

Between 50-60^oC, proteins underwent an intermediate state as shown in (Figure 14 (E)). Interestingly, the melting temperature-curve (the Tm value was determined by previous DSC instrument) of CD spectra of G.h-rTDHY53H/T59I/S63T

was compared to that of G.h-rTDH^WT (Figure 14 (A)). However, G.h-rTDHY53H/T59I/S63T

protein does not transit from a -rich structure into a -rich structure until 75^oC, whereas G.h-rTDH^WT

35

G.h-rTDHY53H/T59I/S63T

has a higher activation energy than that of G.h-rTDH^WT in their phase transition processes. The increase of temperature from 60-95^oC, the

-helix-rich structure content of G.h-rTDH

Y53H/T59I/S63T

was still retained, whereas that of G.h-rTDH^WT was decreased vividly. Interestingly, all G.h-rTDH proteins were not completely denatured even at 95^oC. In 10 mM Kpi buffer (pH 7.0), the temperature-induced conformational change of G.h-rTDHY53H/T59I/S63T

occurs in a two-state manner, while that of G.h-rTDH^WT is in a three-state manner. For other G.h-rTDHY53A/T59B/S63C

mutants, the similar spectrum changes, as that of G.h-rTDHY53H/T59I/S63T

, (in either the far-UV CD spectra or DSC data) were observed in G.h-rTDH^T59I/S63T and G.h-rTDH^Y53H/T59I mutants, from which both of them lost its Arrhenius effect after continuing heating, followed by a rapid cooling treatment (Figure 13 (B)). However, all proteins that have an Arrhenius effect, except for G.h-rTDH^Y53H G.h-rTDH^T59I and G.h-rTDH^S63T mutants, must undergo a transition state prior to the formation of an unfolded state after a thermal denaturation treatment.

In the far-UV CD spectra, the complete collapse of its secondary structure was

FIGURE 13. (A)The CD spectrum of G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

and the G.h-rTDH^WT show three-state and G.h-rTDHY53H/T59I/S63T

show two-state manner when heat the temperature.

37

FIGURE 13. (B) The CD spectrum of G.h-rTDHY53H/T59I/S63T

, G.h-rTDH^Y53H/T59I and G.h-rTDH^T59I/S63T which are deficiency the Arrhenius effect.

FIGURE 13. (C) The CD spectrum of G.h-rTDH^Y53H, G.h-rTDH^T59I and G.h-rTDH^S63T which are process the Arrhenius effect.

39

FIGURE 13. (D) The CD spectrum of G.h-rTDH^WT, G.h-rTDHY53H/T59I/S63T

, and G.h-rTDH^Y53H/S63T. The G.h-rTDH^Y53H/S63T pattern is between G.h-rTDH^WT and G.h-rTDHY53H/T59I/S63T

.

FIGURE 13. (E) The CD spectrum of all mutated G.h-rTDHY53A/T59B/S63C

proteins in 10mM Kpi buffer, pH 7.0. Red box showed the no Arrhenius effect proteins.

41

4.10 Morphology examination and MTT assay for G.h-rTDH

Y53A/T59B/S63C

of the cytotoxicity and cytoviability effect on AGS cells

To investigate the cytotoxicity of purified G.h-rTDHY53A/T59B/S63C

proteins on mammalian cells, the human stomach epithelial cell line, AGS, served as an in vitro model. The morphological change of AGS cells could be visually assessed in the absence or presence of G.h-rTDHY53A/T59B/S63C

exposure for 30 min with 10 μg/mL G.h-rTDHY53A/T59B/S63C

at 26^oC. Among the result of these mutants, the morphology of AGS cells were changed, including the membrane blebbing, the cell detachment, loss of cell cytoplasm with cell shrinkage, and the reduction of nuclei size. min, and the complete dramatic change in the morphology was observed in 30 min. In addition, we used the MTT assay (Figure 16) to analyze the cytoviability when cell was treated with different concentrations of G.h-rTDHY53A/T59B/S63C

(100 μg/mL and different serial dilutions in ten-fold increments) in 1 h at 37^oC. Accordingly, the inhibitory concentration (IC₅₀) value of G.h-rTDH^Y53H/T59I (defined as the drug concentration at which cell growth was inhibited by 50%) at 10 μg/mL was the lowest observed, and the IC₅₀ value of G.h-TDH^Y53H/S63T inhibitory concentration (IC₅₀) at 2

g/mL was the highest assessed from the dose-response curves shown in Figure14.

(A)Control (B) G.h-rTDH^WT (C)G.h-rTDHY53H/T59I/S63T

43

(D) G.h-rTDH^Y53H (E) G.h-rTDH^T59I (F)G.h-rTDH^S63T

FIGURE 14. Cell morphology. AGS cell line was exposed G.h-rTDHY53A/T59B/S63C

10ug/mL in 30min at 26^oC.

(I) G.h-rTDH^T59I/S63T (H) G.h-rTDH^Y53H/S63I

(A) G.h-rTDHY53H,T59I,S63T

S63T

(G) G.h-rTDH^Y53H/T59I

45

FIGURE 15. MTT assay results of various G.h-rTDHY53A/T59B/S63C

mutants. The cytoviability decreased in proportion to different concentrations of G.h-rTDH (100 μg/mL and ten-folded serial dilutions) in 1 h at 37^oC. Data are presented as the means for triplicate experiments. Error bars represent the standard deviations (SD)

4.11 Thioflavin T florescence assay

Thioflavin T fluorescence assay was used to determine the fibrils contents of various TDH mutants, which are generated from a thermal-pretreatment of TDH mutants at either 37

^o

C, 70

^o

C, or 90

^o

C coupled with a rapid cooling, respectively. According to the result of ThT assay, the filber contents of G.h-rTDH

^T59I

, G.h-rTDH

^S63T

, G.h-rTDH

^Y53H/T59I

, G.h-rTDH

^Y53H/S63T

, G.h-rTDH

^T59I/S63T

, and G.h-rTDH

Y53H/T59I/S63T

generated from a 90

^o

C thermal pre-treatment coupled with rapid cooling are higher than that of TDH mutations generated from a 70

^o

C thermal pre-treatment coupled with rapid cooling.

Comparably, the fibrils contents of G.h-rTDH

^WT

and G.h-rTDH

^Y53H

are

similar at either fiber generation from a 90

^o

C thermal pre-treatment or a 70

^o

C thermal pre-treatment, coupled with a rapid cooling, respectively.

FIGURE 16. Thioflavin T florescence assay result. Those curves are presented as the means for triplicate experiments.

G.h-rTDH

^Y53H

G.h-rTDH

^WT

G.h-rTDH

^WT

G.h-rTDH

^Y53H

G.h-rTDH

^T59I

G.h-rTDH

^S63T

G.h-rTDH

^Y53H/T59I

G.h-rTDH

^Y53H/S63T

G.h-rTDH

^T59I/S63T

G.h-rTDH

Y53H/T59I/S63T

37^oC 70^oC 90^oC

47

Chapter 5 Discussion

The data in this thesis showed that the thermostable direct hemolysin ( TDH ) of G. hollisae from BCRC 15890 strain exhibited a distinct biophysical characterization from that of the historically published tdh gene in NCBI data bank, G.

hollisae 9041. First, three distinct amino acid changes, i.e. Tyr53His53, Thr59Ile59, and Ser63Thr63 were observed from the pairwise sequence alignment of these two same sources TDH. Subsequently, seven G.h-rTDH mutants comprising the combination of these three diverse amino acids, i.e. G.h-rTDH^Y53H, G.h-rTDH^T59I, G.h-rTDH^S63T, G.h-rTDH^Y53H/T59I, G.h-rTDH^Y53H/S63T, G.h-rTDH^T59I/S63T, and G.h-rTDHY53H/T59I/S63T

mutants were thus constructed and used to have various biophysical characterizations. Among these mutations, G.h-rTDH^Y53/T59I, G.h-rTDH^T59I/S63T, and G.h-rTDHY53H/T59I/S63T

lost its function of hemolytic activity on erythrocyte cells via first heating at 90^oC for 30 min, coupled with a rapid cooling. The phenomenon of heating inactivation but functional reactivation via further cooling treatment was represented as “Arrhenius effect”, and it is one of significant feature of wild type TDH from G. hollisae. Interestingly, at least mutagenic two amino acid position, i.e G.h-rTDH^Y53H/T59I, G.h-rTDH^T59I/S63T and G.h-rTDHY53H/T59I/S63T

mutants, dramatically influenced their Arrhenius effect, except for G.h-rTDH^Y53H/S63T mutant where a slight enhancement of hemolytic activity on erythrocyte cells was observed.

Moreover, the DSC experimental illustration these G.h-rTDH mutants are in good agreement with the corresponding result from the CD–melting curves of these mutants which were measured by monitoring the temperature change. From DSC data,

the G.h-rTDHY53H/T59I/S63T

protein showed a stable conformation than that of G.h-rTDH^WT protein. Interestingly, no Arrhenius effect proteins involving G.h-rTDH^Y53H/T59I, G.h-rTDH^T59I/S63T, and G.h-rTDHY53H/T59I/S63T

mutants undergo two-state conformational transition change from β-rich structure into a α-rich structure, from the recorded CD spectra during the increasing temperature. Moreover, even at 95^oC those mutant proteins retain the compact secondary structure elements without any complete unfolding, suggesting that the fiber form state existed and caused the proteins to have Arrhenius effect deficiency. The rapid cooling treatment still remains the protein conformation at its fiber form that is a non-toxin form. In contrast, for the single mutation proteins possessing Arrhenius effect, i.e.

G.h-rTDH^Y53H G.h-rTDH^T59I and G.h-rTDH^S63T, the secondary structure collapsed also in a two-state manner. In the G.h-rTDH^Y53H and G.h-rTDH^S63T, when heat temperature above 60^oC their secondary structure complete collapse and protein from β-rich structure into unfold state not through α-rich structure. And the G.h-rTDH^Y53H has more compact than G.h-rTDH^S63T show in figure 16. Interestingly, G.h-rTDH^T59I remained partial α-rich structure when heat to high temperature. Those single mutation proteins when they through the transition state were more unstable than others. Under the heating temperature, the G.h-rTDH^Y53H/S63T protein exhibited a unique characteristic of secondary structure between the data of no-Arrhenius effect proteins and Arrhenius effect proteins. For Arrhenius effect proteins, the intensity of the minima at 218 nm, which represents a β-rich structure, occurs at the low measured temperature that protein exist a native form, decreases the content between 50^oC to 60^oC and completely disappears above at 60^oC and convert to a α-rich structure or a unfolded state. We think those proteins possessing Arrhenius effect at the high

49

However, there is no difference among the observations of the experimental results in the melting curve of CD spectra, SDS-PAGE, and native-PAGE either in stacking or separating gel with all proteins treated with rapid or slow cooling after a first heating.

In the previous study, the crude protein involving TDH from V. parahaemolyticus has similar phenomenon in SDS-PAGE where no band is visualized at 22 KDa. They think there is an inactivating factor to inhibit the TDH hemolysis function and the factor processes proteolytic activity that can digest TDH³⁰. In the other hand, Takashi, F. et. al, suggested that the V. parahaemolyticus TDH process Arrhenius effect phenomena because of the protein structure can recover to its native form and remain the hemolysis function when heated high temperature coupled by a rapid treatment²⁴. However, in our results, those G.h-rTDHY53A/T59B/S63C

remained the secondary structure formation of high temperature when after treatment a rapid cooling or a slower cooling. In this part, the exact reason of the thermal-induced hemolytic activity change as well as its conformational change of G.h-rTDHY53A/T59B/S63C

protein is still unknown. We think the different cooling rate and protein structure stability might influence the protein refolding pathway, thus causing the distinct result. Additionally, the lacking of the detailed structural information in a CD spectrum might also result in the difficulty for interpretation of real-time actual structural features of proteins such as / barrels.

On the other hand, the experimental results demonstrated that various

On the other hand, the experimental results demonstrated that various

在文檔中 探討霍氏格里蒙菌中熱穩定性溶血素之原始型與突變型生化活性與生物物理特性的差異 (頁 30-0)