Journal of Protein Chemistry, Vol. 22, Nos. 7/8, November 2003 (© 2003)
Effect of Ethylene Glycol and Polyethylene Glycol on the Acid-Unfolded State of Trypsinogen Farah Naseem1 and Rizwan Hasan Khan1,2 Received July 9, 2003
Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols. KEY WORDS: Acid-unfolded state; circular dichroism; fluorescence; polyol induced intermediate.
1. INTRODUCTION
states hard to achieve; different methods are employed for the characterization of the folding intermediates and for understanding the different factors leading to the formation and stabilization of these intermediates. A solvent perturbation study is a good approach to evaluate the stabilizing forces of protein structure. One of the interesting cosolvents in this context is polyols and sugars because they have potential protecting effects on various types of protein denaturation. Such cosolvent effects of polyhydric compounds are essentially based on their antagonistic properties to nonpolar groups. The main driving force of protein stabilization by polyols is enhancement of hydrophobic interactions (Gekko, 1981; Gekko and Morikawa, 1981; Gekko and Timasheff, 1981; Xie and Timasheff, 1997). In this report, we show that polyols (ethylene glycol and polyethylene glycol) at high concentrations induce secondary structure in acid-induced unfolded structure of trypsinogen. These polyols also show some stabilizing effect on the tertiary structure of the protein at low pH. Trypsinogen belongs to the family of serine proteases. Pancreatic trypsin is produced in its zymogen form, trypsinogen, which is converted into trypsin by
In recent years, there has been growing recognition of the importance of the compact denatured states of proteins. Characterization of these structures and the factors involved in their stability would provide important insight into the interactions responsible for their formation as well as their role in protein folding (Prajapati et al., 1998). The development of a broad range of techniques has led to the identification and characterization of stable intermediates in several proteins (Barrick et al., 1994; Bongiovanni et al., 2001; Kumar et al., 1994; Matouschek et al., 1992). Recently, a number of equilibrium intermediates have been detected and characterized. In our earlier communications, we have reported the molten globule states in proteins such as ␣-chymotrypsinogen-A, stem bromelain, glucose oxidase (Khan et al., 2000; Gupta et al., 2003; Haq et al., 2002; Haq et al., 2003) and fetuin (Naseem et al., 2003). The high cooperativity and complexity of the protein folding process makes the characterization of conformational transitions and equilibrium intermediate 1
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India. 2 To whom correspondence should be addressed. E-mail: rizwanhkhan@ hotmail.com;
[email protected]
3
Abbreviations: EG, ethylene glycol; PEG, polyethylene glycol; CD, circular dichroism; GnHCl, guanidine hydrochloride.
677 0277-8033/03/1100-0677/0 © 2003 Plenum Publishing Corporation
678 the action of another pancreatic enzyme, enterokinase, that splits off the N-terminal end of trypsinogen. Trypsinogen consists of 229 amino acid residues with a molecular weight of 24 kDa. Bovine trypsinogen contains four tryptophans at positions 51, 141, 215, and 237. All tryptophans are highly buried: Trp 51, 96%; Trp 141, 98.7%; Trp 215, 80%; and Trp 237, 80% (Otlewski et al., 1996). 2. MATERIALS AND METHODS 2.1. Materials GnHCl3 was purchased from Sigma Chemical Co. (St. Louis, MO). Ethylene glycol and polyethylene glycol (PEG)-400 were obtained from Qualigens (Mumbai, India). All other reagents and buffer components used were of analytical grade. 2.2. Methods Acid-induced unfolding was carried out by taking the protein in 10 mM glycine-HCl buffer, pH 1, and extensively dialyzing it against the same. Concentration of different polyols (i.e., EG and PEG-400) was varied from 0%–90% (v/v) in samples containing 0.2 mg/ml protein. 2.2.1. Spectrophotometric Measurements The protein concentration was determined on a Hitachi U-1500 (Tokyo, Japan) spectrophotometer using the molar absorption coefficient of ε = 33360 M−1 cm−1 at 280 nm (Vincent and Lazdunski, 1976) and by the method of Lowry et al. (1951). 1-anilino-8-naphthalene sulfonic acid (ANS) concentration was also determined spectrophotometrically using a molar absorption coefficient of 5000 M−1 cm−1 at 350 nm (Khurana and Udgaonkar, 1999). The molar ratio of protein to ANS was 1:100. 2.2.2. CD Measurements CD measurements were recorded with a Jasco spectrophotometer (model J-720) (Tokyo, Japan) equipped with a microcomputer precalibrated with D-10camphosulfonic acid. All the measurements were carried out at 25°C, and each spectrum was recorded as an average of two scans. The near-UV spectra were recorded in the wavelength region of 250–300 nm in a 10-mm pathlength cuvette. The far-UV spectra were recorded in the
Naseem and Khan wavelength region of 200–250 nm in a 1-mm path-length cuvette. The protein concentration was 0.2 mg/ml for both near and far-UV CD studies. Results are expressed as mean residue ellipticity (MRE) values as reported in our earlier communications (Khan et al., 2000; Gupta et al., 2003; Haq et al., 2002; Haq et al., 2003): MRE = obs (mdeg)/10 × n × Cp × l where obs is the CD in millidegree, n is the number of amino acid residues, l is the path-length of the cell in cm, and Cp is the molar fraction.
2.2.3. Fluorescence Measurements Fluorescence measurements were carried out on a Shimadzu Spectrofluorometer (model RF-540) (Kyoto, Japan) equipped with a data recorder DR-3. For intrinsic tryptophan fluorescence, the excitation wavelength was set at 280 nm and emission spectra recorded in the range of 300–400 nm. Binding of ANS to acid-unfolded trypsinogen was studied by exciting the dye at 380 nm, with emission range being 400–600 nm. Protein concentration used was 0.05 mg/ml.
3. RESULTS AND DISCUSSION 3.1. CD Studies The effect of EG and PEG-400 on the acid-unfolded structure of trypsinogen at pH 1 was studied by means of both far- and near-UV CD measurements. Figure 1(a) shows the far-UV CD spectra of acid-unfolded trypsinogen treated with different concentrations of EG (0–90% v/v). As can be seen from the figure, the spectrum of acid-denatured trypsinogen in absence of EG shows an unordered structure with a CD band at 204 nm (a sharp trough near 200 nm is characteristic of unordered structure). At very low concentration (10% v/v), EG has very little effect on the unordered structure. In presence of 30% and 50% v/v EG, protein spectra show some features of ordered secondary structure whereas at EG concentrations of 70%, 80%, and 90%, induction of secondary structure is evident. The changes in MRE values and CD minima with varying concentrations of EG are summarized in Table 1. As can be seen from Fig. 1(b) and Table 2, with increase in concentration of PEG there is continuous increase in negative MRE value at 217 nm. Up to 30% PEG, protein remains in unordered structure whereas at 50% PEG, it adapts a similar conformation as in the presence of 90% EG. Interestingly, further
Effect of EG and PEG on the Acid-Unfolded State of Trypsinogen
679
Fig. 1. (a) Far-UV CD spectra of trypsinogen at pH 1 and at varying concentrations of ethylene glycol, 10%–90% (v/v): (-----) acid-unfolded state (i.e., at pH 1); (—䊊—) 10% EG; (—䉭—) 30% EG; (— ..—) 50% EG; (—×—) 70% EG; (—.—) 80% EG; and (......) 90% EG. Protein concentration was 0.2 mg/ml. Spectra were recorded as mean residue ellipticity in the wavelength 200–250 nm at 25°C. (b) Far-UV CD spectra of trypsinogen at pH 1 and at varying concentrations of polyethylene glycol 10%–80% (v/v): (-----) acid-unfolded state (i.e., at pH-1); (—䊊—) 10% PEG; (—䉭—) 30% PEG; (—..—) 50% PEG; (—×—) 70% PEG; and (—.—) 80% PEG. Conditions were similar as those for Fig. 1(a).
increase in concentration (60%–80% v/v) led to the formation of -structure with a strong negative band at 217 nm. Presence of predominant -structure in trypsinogen in the form of two -barrels has been reported by Otlewski et al. (1996). The effect of the two polyols on the tertiary structure of acid-denatured trypsinogen was investigated by near-UV CD (Fig. 2). As previously discussed, far-UV CD studies show the transition of trypsinogen at acidic pH from unordered to an ordered state. Interestingly, some amount of stabilizing effect of these two polyols is also observed on the tertiary structure. Native trypsinogen shows negative bands with three considerable maxima at 256 nm, 265 nm, and 295 nm and a minimum at 272 nm. These Table 1. Far-UV CD Data at Different Minima Values in Presence of EG
Subject Acid-unfolded trypsinogen 10% EG 30% EG 50% EG 70% EG 80% EG 90% EG
MRE at 204 nm
MRE at 208 nm
−23,000
—
−23,000 — — — — —
— −17,500 −20,000 −21,000 −17,000 −21,000
characteristic CD bands are disrupted at low pH (Fig. 2). In presence of high concentrations of EG and PEG, the spectra of acid-denatured trypsinogen shift toward the negative MRE values (Table 3) and show resemblance to the spectrum of native trypsinogen. 3.2. Fluorescence Studies Intrinsic as well as extrinsic fluorescence studies were performed to see the effect of EG and PEG on the conformation of acid-unfolded trypsinogen. The ability of PEG to bind the hydrophobic patches and absorption by EG and PEG alone were also taken into account, and we report here the subtracted fluorescence spectra.
Table 2. Far-UV CD Data at Different Minima Values in Presence of PEG
MRE at 222 nm −8500 −9500 −11,000 −12,000 −16,000 13,000 −18,500
CD, circular dichroism; EG, ethylene glycol; MRE, mean residue ellipticity.
Subject Acid-unfolded trypsinogen 10% PEG 30% PEG 50% PEG 70% PEG 80% PEG
MRE at 204 nm
MRE at 217 nm
−23,000
−11,000
−19,000 −16,500 — — —
−11,500 −13,000 −18,000 −21,500 −23,000
CD, circular dichroism; PEG, polyethylene glycol; MRE, mean residue ellipticity.
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Naseem and Khan
Fig. 2. Near-UV CD spectra of trypsinogen: (—) native state at pH-7; (------) acid-unfolded state at pH 1; (—.—) in presence of 80% PEG v/v; and (......) 90% EG v/v. Protein concentration was 0.5 mg/ml and spectra were recorded as mean residue ellipticity in the wavelength range 250–300 nm at 25°C.
Tryptophan fluorescence of acid-unfolded trypsinogen increased with increasing concentrations of EG and PEG. Up to 20% of both EG and PEG, increase in fluorescence intensity was to the same extent, but at polyol concentrations greater than 20%, increase was more significant in the case of PEG (Fig. 3). Figure 4 shows that at pH 1, when the protein is in polar environment, the tryptophan residues, which are buried in the native structure, get exposed due to acid unfolding; this is evident by low fluorescence intensity and redshift in max of approximately 2 nm as compared to the native spectrum, but the redshift is less than the protein in presence of GnHCl (i.e., at pH 1, protein is not completely denatured). Addition of PEG to the acid-unfolded state increases the hydrophobic interactions (Gekko, 1981; Table 3. Summary of Near-UV CD Data of Trypsinogen
Subject Native trypsinogen Acid-unfolded trypsinogen 80% PEG 90% EG
MRE at 256 nm
MRE at 265 nm
MRE at 272 nm
MRE at 295 nm
−20
−40
−100
−70
+80
+60
+50
+100
−20 −40
−40 −30
−70 −50
+20 +30
CD, circular dichroism; EG, ethylene glycol; PEG, polyethylene glycol; MRE, mean residue ellipticity.
Fig. 3. Relative tryptophan fluorescence as a function of (▲) %EG and (●) %PEG (v/v). Spectra were recorded at 25°C with excitation wavelength of 280 nm. Protein concentration used was 0.05 mg/ml.
Gekko and Timasheff, 1981; Gekko and Morikawa, 1981; Xie and Timasheff, 1997) and the acid-unfolded state of trypsinogen which showed unordered structure acquires a conformation with ordered secondary structure. We speculate that the tryptophan residues in this polyol-induced state are somewhat exposed to the environment in contrast to the highly buried tryptophan residues in the native protein. Thus, as the PEG concentration is increased, the partially exposed tryptophan residues face more and more nonpolar environment, explaining the blueshift in max and enhancement in tryptophan fluorescence. Similar spectra were obtained in the case of EG, but the effect was less pronounced (Fig. 3). The overall effect of polyols on the tertiary structure of acid-unfolded protein was found to be stabilizing, but different from the native state (Fig. 2). The change in the secondary structure of the acid-unfolded trypsinogen brought about by high concentrations of EG and PEG is clearly visible in Fig. 1. The intermediate state induced by polyols is further confirmed by extrinsic fluorescence studies. ANS fluorescence studies showed that up to 60% EG, there was low ANS binding whereas at higher concentrations (70%–90% v/v), a slight increase in
Effect of EG and PEG on the Acid-Unfolded State of Trypsinogen
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Fig. 4. (—) Fluorescence emission spectra of native trypsinogen; (—.—) acid-unfolded state at pH-1; (-----) in presence of 80% PEG v/v; and (......) in presence of 6 M GnHCl. An increasing trend in the tryptophan fluorescence was observed with increasing concentrations of PEG; spectra not shown for the sake of clarity.
fluorescence intensity was observed. Similarly, in the case of PEG, ANS showed low binding at low concentrations (up to 40%), but a marked increase in fluorescence intensity was observed above 40% (Fig. 5). Maximum ANS binding at high concentrations of both EG and PEG (Fig. 5), low ANS binding at pH 1, and negligible binding at pH 7 and in presence of GnHCl (spectra not shown) indicate the presence of the abovementioned intermediate having properties similar to that of the molten globule (MG) state. This intermediate state has some secondary structure as well as some tertiary contacts as indicated by CD studies, but the structure is different from the native state. Thus, the above results indicate that both EG and PEG have similar behavior toward the acid-unfolded trypsinogen. They stabilize the unfolded protein by inducing secondary structure, but in all the cases the effect of PEG is more pronounced as compared to EG; this is because PEG has a larger molecular weight and greater number of OH groups. The effect of polyols increase with increasing concentration and number of OH groups (Kamiyama et al., 1999). Formation of secondary structure in acid-denatured trypsinogen in presence of polyols can be attributed to the enhanced hydrophobic interactions able to overcome electrostatic repulsions among charged side-chains, as stated by Kamiyama and colleagues in their studies on the effect of sorbitol (a polyol) on cytochrome C (Kamiyama et al., 1999).
Fig. 5. Relative ANS fluorescence as a function of (▲) %EG and (●) %PEG. Excitation wavelength was 380 nm. Other conditions were similar as mentioned for Fig. 3.
Similar results were reported by Bongiovanni et al., 2002, on the effect of glycerol on acid-denatured cytochrome C. Their results indicated that glycerol induces collapse of unfolded protein into a compact state at very high concentrations (90%). Similarly, in this study it is shown that treating acid-unfolded trypsinogen with high concentrations of ethylene glycol and polyethylene glycol result in a transition from an unordered structure to an intermediate state having ordered secondary structure as well as some tertiary contacts, but different from the native state.
4. CONCLUSIONS On the basis of several studies performed to date, it has been established that high-molecular-weight polyols stabilize protein structure. In our studies, we have shown that even ethylene glycol and polyethylene glycol-400 are capable of inducing secondary structural elements in acid-unfolded trypsinogen. Interestingly, some amount of stabilizing effect of these two polyols was also observed on the tertiary structure of the protein.
682 ACKNOWLEDGMENTS Facilities provided by Aligarh Muslim University are gratefully acknowledged. The authors are also thankful to the Department of Science and Technology (DST) and the Fund for the Improvement in Science and Technology Infrastructure (FIST) for providing lab facilities.
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