|UREA AND THERMAL UNFOLDING OF COLD-ADAPTED ATLANTIC COD (Gadus morhua) AND PORCINE PEPSINOGENS
Department of Food Science, Faculty of Agrotechnology and Food Science
Kolej Universiti Sains dan Teknologi Malaysia
21030 Mengabang Telipot, Kuala Terengganu, Terengganu
R.K. OWUSU APENTEN
Department of Food Science
The Pennsylvania State University
College of Agriculture Sciences
111 Borland Laboratory, University Park, PA 16802
Abstract: - The reversible unfolding reactions for Atlantic cod pepsinogen and porcine pepsinogen were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. Studies were performed at pH 6-8 to avoid activation and autolysis of both pepsinogens. Urea and thermal unfolding studies showed that the conformation of cod pepsinogen is less stable than porcine pepsinogen, A slightly lower value of Cp for porcine pepsinogen indicates that porcine pepsinogen has a lower hydrophobicities compared to cod pepsinogen. This implies that elestrostatic and hydrogen bonding interactions is more important than hydrophobic interactions in stabilising the native conformation of porcine pepsinogen. Both cod and porcine pepsinogens had Ho > -TSo. A predominant enthalpic contribution suggests that stabilisation is mainly by electrostatic and/or hydrogen bond interactions (as compared to hydrophobic interactions) for both pepsinogens.
Key-Words: - pepsinogen, cod, porcine, unfolding, urea, thermal, conformational stability
Pepsin refers to a series of gastric proteinases secreted as inactive precursors (pepsinogen) and which are active at acidic pH.
Pepsin and pepsinogen from fish have been purified from various species such as Greenland cod, bluefin tuna, polar cod, sardine, hake and finfish orange roughy [1,2,3,4,5,6,7,8,9].
Cold-adapted proteinases from fish have some unique properties such as, a lower inactivation temperature, high molecular activity at low temperature and an increased ability to catalyse the hydrolysis of native protein substrates when compared with proteinases from mammals, thermophilic organisms and plants [10,11,12]. Highly active and thermolabile fish proteinases have potential applications in the commercial manufacture of cheese and protein hydrolysates [11,12,13 ]. The combination of high catalytic activity and low thermal stability is thought to be a consequence of the greater molecular flexibility of cold-adapted enzymes [14,15].
The isolation, characterization, and possible applications of cod digestive proteinases have been extensively studies [16,13,17,18,19,12]. For the time being, only a few reports concerning the irreversible inactivation of fish pepsin and/or pepsinogen have been published [1,20,21]. However, no studies has been reported on the reversible unfolding (or conformational stability) of fish pepsin or pepsinogen either by urea or thermal unfolding.
The conformational stability of a globular protein may be defined as the free energy change for the reaction:
native Û denatured (1)
The thermodynamic or conformational stability of a protein can be measured as a function of temperature or denaturant concentration. Reversible unfolding studies allow measurement of conformational or thermodynamic stability. The methods of protein unfolding reaction thermodynamics involves a number of assumptions [22,23,24,25,26,27,28] such as, the unfolding proceeds via a two-state mechanism and that the free energy may be obtained from the apparent values in the presence of denaturants by extrapolation to zero denaturant concentration [23,27,29,30,31].
For proteinases, it is important to perform unfolding studies under conditions where autolysis does not occur. In this study, the unfolding of cod pepsinogen and porcine pepsinogen was studied as a function of temperature and urea concentration in the pH range of 6-8. Studies were performed at pHs above 5 to avoid activation and autolysis of both pepsinogens.
The aim of this study is to compare the conformational stability of cold-adapted pepsinogen from Atlantic cod (Gadus morhua) with that of porcine pepsinogen.
2. Materials and Methods
Whole fresh Atlantic cod (Gadus morhua) were supplied by a wholesaler in Bridlington (Humberside, UK). Atlantic cod pepsinogen was purified to homogeneity by a combination of affinity chromatography on hemoglobin-Sepharose and gel filtration with a 83- fold purification and a yield of 29% . Porcine pepsinogen and Trizma basewere supplied by Sigma Chemical Co., Poole (UK). Urea was puris grade (Fluka Chemical Ltd., Glossop, UK). All other chemicals were of AnalaR grade from the British Drug Houses (BDH) Ltd. (UK).
2.1 Determination of fluorescence emission spectrum for native, thermally-unfolded and urea-unfolded pepsinogen
The instrument used to monitor the unfolding of both pepsinogens was a fluorescence spectrophotometer (Perkin Elmer 203, Beaconsfield, Bucks, UK) fitted with a digital output and a thermoregulated twin cuvette holder. Temperature control was by means of a circulating water bath.
A suitable fluorescence emission wavelength for fluorimetry measurements were determined after plotting a fluorescence emission spectrum (fluorescence intensity versus emission wavelength) of native, thermally-unfolded and urea-unfolded for cod pepsinogen and porcine pepsinogen. The emission wavelength chosen for thermal unfolding was the one that would reflect the largest difference between the fluorescence intensity of native and thermally-unfolded pepsinogen. Whilst for urea unfolding, the suitable emission wavelength was the one that would reflect the largest difference between the fluorescence intensity of native and urea-unfolded pepsinogen
2.2 Thermal unfolding of Cod pepsinogen (CPG) and porcine pepsinogen (PPG)
To determine the thermal unfolding profiles, each pepsinogen was heated in a quartz cuvette at 5-85oC. The fluorescence measurements were made using an excitation and emission wavelengths of 295 nm and 335 nm for CPG and 295 nm and 340 nm for PPG. Sample temperatures were monitored using a thermocouple immersed in a "reference" cuvette situated adjacent to the sample cuvette. After each experiment heated enzyme samples were cooled to room temperature in order to discover whether thermal transitions were reversible.
2.3 Urea unfolding of CPG and PPG
The fluorescence measurements were made using an excitation and emission wavelengths of 295 nm and 320 nm for CPG and 295 nm and 345 nm for PPG. To determine the unfolding profile as a function of urea concentration, same amount of pepsinogen (0.2 ml in 0.05 M Tris-HCl buffer, pH 8.0) were added to different concentrations of urea (prepared from a 10 M urea stock solution). The mixtures were allowed to stand for 3 hr at 25oC to reach equilibrium
After each experiment, the reversibility of unfolding was checked by diluting the urea unfolded sample with appropriate volumes of buffer, and measuring fluorescence changes at lower urea concentration.
2.4 Stability estimates from urea and thermal unfolding profiles
Urea and thermal unfolding profiles were calculated from fluorescence measurements (see below) with the aid of spread sheets (Microsoft Excel, Microsoft Corp. USA). The algorithm used in conjunction with the spread sheet is described below (equation 2-4).
2.4.1 Analysis of unfolding profiles
For both urea and thermal unfolding profiles, the fraction of unfolded enzyme (Fu) and unfolding equilibrium constant (Ku) were determined from the changes in fluorescence emission using equation (2) and (3):
Fu = (yN - yobs) / (yN - yD) (2)
Ku = (1- Fu) / Fu = (yN - yobs) / (yobs - yD) (3)
where yobs is the observed fluorescence intensity while yN and yD represent the fluorescence intensity of the folded and unfolded states respectively. yN and yD are assumed to show a linear dependence on urea concentration and temperature (29,31,32].
2.4.2 Conformational stability towards urea unfolding
From the graph of free energy change (i.e. DG = -RT ln Ku , where R is the gas constant and T is the absolute temperature) versus urea concentration, a straight line relation exits according to equation 4
DG = DG(H2O) - m[urea] (4)
where DG(H2O) is the free energy change at zero concentration of urea and m (kJMol-1M-1) is the slope of the plot (25,22,20]. The mid point concentration for urea unfolding curve, [urea]1/2 can be obtained from the plot of fraction unfolded vs. urea concentration or from equation (4), i.e. when DG is zero the [urea]1/2 value is given by DG(H2O) / m
2.4.3 Conformational stability towards thermal unfolding
The temperature dependence of DG can be expressed as a second order polynomial [22,33,34,35,36].
DG = A + B T + C T2 (5)
The constants A, B, and C were determined by non-linear regression of DG versus T (in Kelvin) data. A, B, and C are a function of the enthalpy ( DH) , entropy (DS) and heat capacity change (DCp) for enzyme unfolding according to equation 6.
DH = A - C T2 (6a)
DS = - (B - 2CT) (6b)
DCp = 2 CT (6c)
3. Results and Discussion
3.1 Conformational stability of CP and PP
The conformational stability of porcine pepsin and pepsinogen towards heat and/or urea unfolding have been extensively studied. Conformational stability studies involving porcine pepsin and pepsinogen is normally carried out either with pepsinogen or pepsin (preferably inhibited-pepsin) at pH 6-8. However, no conformational stability studies has been reported on fish pepsin and pepsinogen. Studies were carried out in the hope of shedding further light on kinetic stability of cod pepsin and CPG.
3.1.1 Conformational stability towards urea unfolding
A suitable fluorescence emission wavelength for monitoring urea and thermal unfolding was determined from the fluorescence spectra (Figure 1A and 1B).
The fluorescence excitation wavelength was fixed at 295 nm, at which only tryptophan residues were excited. The suitable wavelength was chosen as the wavelength where the greatest difference in fluorescence emission intensity was noted between the native and unfolded pepsinogen.
Figure 1A and 1B show the fluorescence emission spectrum for native and urea unfolded CPG and PPG at pH 7.0, respectively. max for native, urea unfolded and thermally unfolded CPG occured at 335, 345 and 335, respectively. max for native, urea unfolded and thermally unfolded PPG were observed at 340, 345 and 330, respectively. The value of max depends on exposure of tryptophan residues in the protein to the solvent [37,38]. Urea unfolding was associated with higher max for both CPG and PPG, thus indicating the increase in the exposure of tryptophan residues to the solvent. Thermal unfolding did not change the max of CPG, but decreased the max of PPG.
This suggests that the exposure of tryptophan residues to the solvent was not altered in CPG, but decreased in PPG. The decrease of tryptophan exposure may be caused by aggregation upon heating at pH 7.0.
Figure 1A: Fluorescence emission spectrum for CPG and the same concentration of enzyme in 7.87 M urea (urea unfolded) or after heating to 85oC (thermally unfolded).
Figure 1B: Fluorescence emission spectrum for porcine pepsinogen and the same concentration of enzyme in 7.87 M urea (urea unfolded) or after heating to 85oC (thermally unfolded).
Figure 2A and 2B show the fluorescence intensity versus urea concentration for CPG and PPG.
Figure 2A: Effect of urea on the fluorescence intensity of cod pepsinogen at 25oC.
Figure 2B: Effect of urea on the fluorescence intensity of porcine pepsinogen at 25oC.
Urea and GdmCl are the most commonly used denaturants for studying unfolding. They tend to increase protein solubility and to interact preferentially with the protein surface, thus appearing to be bound .
A normalized unfolding profile could not be generated for CPG because of the difficulties in resolving its QN. The normalized profile for PPG is shown in Figure 3. One can see that the profile was not very smooth. This may indicate a possibility that the unfolding pathway was not a two-state model under the condition studied.
Figure 4 shows the plot of first derivative (fluorescence intensity versus urea concentration) versus urea concentration for both pepsinogens. The plot shows that the unfolding transition starts at lower concentration (slightly above 0 M) for CPG as compared to PPG (1.67 M). Earlier studies on urea unfolding of PPG by difference absorption spectra seemed compatible with a simple two-state mechanism . In contrast, kinetic experiments of PPG with various urea concentration revealed the presence of high concentration of one or more transient intermediates on the folding pathway [40,41]. It is possible that fluorimetry was more sensitive than difference absorption spectra in detecting changes during unfolding. Wada et al.  reported that the GdmCl unfolding of PPG was a multi-step transition by four different spectroscopic dimensions (absorbance, CD, fluorescence and light scattering intensity).
Figure 3: The normalized unfolding profile for porcine pepsinogen.
Figure 4: The plot of first derivative versus urea concentration for cod and porcine pepsinogens.
3.1.2 Conformational stability towards thermal unfolding
Thermal unfolding of CPG and PPG was examined at pH 6, 7 and 8. Figure 5A and 5B shows the fluorescence intensity of CPG and PPG as a function of temperature at pH 6-8, while Figure 6A and 6B present their normalized unfolding profiles. Mateo and Privalov  and Privalov et al.  reported that the thermodynamics of the thermal unfolding of PPG (using DSC) could be discussed using a two-state model.
Both CPG and PPG were more stable at pH 6 than at pH 7 or pH 8.
Figure 5A: Effect of temperature on the fluorescence intensity of cod pepsinogen at pH 6, 7 and 8.
Figure 5B: Effect of temperature on the fluorescence intensity of porcine pepsinogen at pH 6, 7 and 8.
Figure 6A: The normalized thermal unfolding profiles of cod pepsinogen at pH 6, 7 and 8.
Figure 6B: The normalized thermal unfolding profiles of porcine pepsinogen at pH 6, 7 and 8.
Tm values shown in Table 1 shows that PPG was more stable towards thermal unfolding than CPG in the pH range of 6-8. Tm values for PPG from this studies were close to that of Privalov et al. .
Table 1: Values for Tm (oC) for CPG and PPG heat unfolding at different pHs
*from Privalov et al. 
Data are the average of triplicates. The relative
error associated with Tm was about 5%.
The results for heat unfolding parameters at pH 7 determined for CPG and PPG using Gibbs-Helmholtz equation are summarized in Table 2.
Table 2: Comparison of the conformational stability parameters for CPG and PPG at pH 7
Data are the average of duplicates. The average error associated with these calculation was 10%.
The greater free energy change at 25oC (Go) of PPG as compared to CPG shows that PPG is more stable than CPG towards thermal unfolding. Comparison of Hm values indicates that unfolding of CPG appears to be a less endothermic reaction compared to the unfolding reaction of PPG. The entropy changes for unfolding (S and Sm) were lower for CPG. Cp is proportional to the non-polar surface area exposed to water by unfolding; that is to the hydrophobicities of the protein interior . CPG had higher Cp compared to PPG. This indicates that CPG may have higher hydrophobicities compared to PPG. Fish pepsinogens appear to have higher hydrophobicity compared to PPG. Lower Cp for PPG implies that the contribution of electrostatic and/or hydrogen bonding interactions is more important than hydrophobic interactions in stabilizing the conformation of PPG. This results is in contrast to that of bovine trypsin whereby hydrophobic interactions play an important role in stabilizing its conformational structure compared to electrostatic and hydrogen bonding interactions .
It is useful to compare H and S values. For CPG and PPG, Ho > -TSo. A predominant enthalpic contribution suggests that stabilisation is mainly by electrostatic and/or hydrogen bond interactions (as compared to hydrophobic interactions) for both pepsinogens; which is also the case for cod trypsin . Electrostatic and/or hydrogen bond interactions are more important than hydrophobic interactions in stabilizing the conformational structure of cod trypsin and CPG. This conclusion agrees with the theory that cold-adapted enzymes have flexible structure because they are stabilized by weak interactions such as electrostatic and hydrogen bonding interactions .
In conclusion, the results of this study suggests that the native conformation of CPG is less heat stable compared to the native conformation of PPG at room temperature.
Further studies, involving a wider range of solvent conditions, are necessary in order to produce a better appreciation of the molecular strategies for cold-temperature adaptation in Atlantic CPG.
M.A.A acknowledges financial support from the Malaysian government and Universiti Sains Malaysia for completing this research.
 Squires, J., Haard, N.F and Feltham, L.A.W. Gastric proteases of Greenland cod (Gadus ogac). I. Isolation and kinetic properties. Biochem. Cell Biol. Vol. 64, 1986. 205-214.
 Tanji, M., Kageyama, T. and Takahashi, K. Tuna pepsinogens and pepsins: Purification, characterization and amino acid sequences. Eur. J. Biochem. Vol. 177, 1988, 2511-259.
 Brewer, P., Helbig, N. and Haard, N.F. Atlantic cod pepsin – Characterization and use as a rennet substitute. Can. Inst. Food Sci. Technol. Vol. 17, 1984, 38-43.
 Aruchalam, K. and Haard, N.F. Isolation and characterization of pepsin from polar cod (Boreogadus saida). Comp. Biochem. Physiol. Vol. 80B, 1985, 467-473.
 Gildberg, A. and Raa, J. Purification and characterization of pepsins from Atlantic cod (Gadus morhua). Adv. Exp. Med. & Biol. Vol. 306, 1983, 107-110.
 Noda, M. and Murakami, K. Studies on proteinases from the digestive organs of sardine I. Purification and characterization of three alkaline proteinases from the pyloric caeca. Biochim. Biophys. Acta Vol. 658, 1985, 27-34.
 Sanchez-Chiang, L. and Ponce, O. Gastricsinogens and gastricsins from Merluccius gayi. Purification and properties. Comp. Biochem. Physiol. Vol. 68B, 1981, 251-257.
 Xu, R. A.., Wong, R. J., Rogers, M. L. Aand Fletcher, G. C. Purification and characterization of acidic proteases from the stomach of the deepwater finfish orange roughy (Hoplostethus atlanticus). J. Food Biochem. Vol. 20(1), 1996, 31-48.
 Amiza, M. A. and Owusu Apenten, R. K. A single-step purification of gastricsin proteinase from Atlantic cod (Gadus morhua). Online J. Bio. Sci. Vol. 2 (9), 2002, 591-595.
 Simpson, B.K. and Haard N.F. Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties. Comp. Biochem Physiol., Vol. 79B, 1984, 613-622.
 Simpson, B.K. and Haard N.F. Cold adapted enzymes from fish. In: Food Biotechnology, D. Knorr (Ed.), Marcel Dekker, Inc., New York, USA, 1987, pp 495-527.
 Simpson, B.K., Simpson, M.V. and Haard N.F. Properties of trypsin from pyloric ceca of Atlantic cod (Gadus morhua). J. Food Sci., Vol. 55, 1990, 959-961.
 Haard, N.F. A review of proteolytic enzymes from marine organisms and their application in the food industry. J. Aquat. Food Product Technol. Vol. 1, 1992, 17-35.
 Hultin, H. O. Enzymes from organisms acclimated to low temperatures. In Enzymes: The interface between technology and Economics. Danehy, J. D. & Wolnak B. (eds.), Marcel Dekker, New York, 1978, pp 161-178.
 Hochachka, P. W & Somero, G. N. Biochemical Adaptation. Princetown University Press, Surrey UK. 1984.
 Asgeirsson, B., Fox, J.W. and Bjarnason, J.B. Purification and characterisation of trypsin from the poikilotherm Gadus morhua. Eur. J. Biochem. Vol. 180, 1989, 85-94.
 Bjarnason, J.B. and Asgeirsson, B. Psychrophilic proteolytic enzymes from Atlantic cod: their characteristics and applications. Genetic Engr. Biotechnol. Vol. 13, 1993, 31-39.
 Raae, A. J. & Walther, B. T. Purification and characterisation of chymotrypsin, trypsin and elastase like proteinases from cod (Gadus morhua). Comp. Biochem. Physiol. Vol. 93B, 1989, 317-324.
 Simpson, B.K., Smith, J.P. and Yaylayan, V. Kinetic and thermodynamic characteristics of a digestive proteases from Atlantic cod (Gadus morhua). J. Food Biochem, Vol. 55, 1989, 959-961.
 Twining,S.S., Alexander, P.A., Huibregtse, K. and Glick, D.M. A pepsinogen from rainbow trout. Comp. Biochem. Physiol. Vol. 75B, 1981, 109-112.
 Amiza M.A. and Owusu Apenten, R. K. Thermal inactivation parameters for cod pepsin, cod pepsinogen and porcine pepsin. 2002 WSEAS International Conferences MCBC, MCBE, ICAMSL, ICAI, Puerto De La Cruz, Tenerife, Spain, Dec. 19-21, 2002. (N.Mastorakis (Greece), Meng Joo Er (Singapore), Carlos D'Attelis (Argentina) Editors)
 Brandt, J.F. The thermodynamics of protein denaturation 1. The denaturation of chymotrypsinogen. J. Am. Chem. Soc. Vol. 86, 1964, 4291-4301.
 Aune, K.C. & Tanford, C. Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. II. Dependence on denaturant concentration at 25 oC. Biochemistry Vol. 8, 1969, 4586-4590.
 Hermans, J. Methods for the study of reversible denaturation of proteins and interpretation of data. Meth. Biochem. Anal., Vol. 13, 1965, 81-111.
 Pfeil, W. & Privalov, P. L. Conformation changes in proteins in Biochemical Thermodynamics, Jones N. M. (ed.). Elsevier Sci. Pub. Co. Oxford 1979. pp 75-115.
 Privalov, P. L. Stability of proteins. Small globular proteins. Adv. Protein Chem. Vol. 33, 1979, 161-241.
 Schellman, J. A. and Hawkes, R. B. Protein Folding: Proc. 28th German Biochemical Society, Regensburg, R. Jaenicke (Ed.), Elsevier/North-Holland Biomedical Press, Amsterdam, 1980, pp 331-341.
 Creighton, T. E. Protein folding. Biochem. J. Vol. 270, 1990, 1-16.
 Pace, C.N. Measuring and increasing protein stability. Trends Biotechnol., Vol. 8, 1990, 93-97.
 Becktel, W. J. & Schellman, J. A. Protein stability curves. Biopolymers Vol. 26, 1987, 1859- 1877.
 Santoro, M. M. and Bolen, D. W. Unfolding free energy changes determined by the linear extrapolation method. I. Unfolding of phenylmethylsulfonyl alpha-chymotrypsin using different denaturants. Biochemistry Vol. 27, 1988, 8063-8068.
 Permyakov, E. A. & Burhstein, E. A. Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence. Biophys. Chem. Vol. 19, 1984, 265-271.
 Brandt, J. F. The nature of the complexities in the ribonuclease conformational transition and implications regarding clathirating. J. Am. Chem. Soc. Vol. 87, 1965, 2759-60.
 Shiao, D. F., Lumry, R. & Fahey, J. Studies of the chymotrypsinogen family of proteins. XI. Heat capacity changes accompanying reversible thermal unfolding of proteins. J. Am Chem. Soc. Vol. 93, 1971, 2024-35.
 Cantor, C.R. & Schimmel, P.R. Biophysical Chemistry III: The behaviour of Biological Macromolecules. W. H. Freeman & Co. (Pub), San Francisco 1980, pp. 1075-1107 & A-17.
 Ragone, R., Facchiano, F., Cacciapouti, G., Porcelli, M, & Colonna, G. Effect of temperature on the propylamine transferase from Sulfolobus solfataricus an extreme thermophilic archaebacterium. 2. Denaturation and structural stability. Eur J. Biochemistry Vol. 204, 1992, 483-490.
 Teale, F. W. J. Ultraviolet and fluorescence of protein in neutral solutions. Biochem. J. Vol. 76, 1960, 381-388.
 Burhstein, E. A., Vedenskina, N. S. and Ivkona, M. N. Fluorescence study of actin. Photochem. Photobiol. Vol. 18, 1973, 263-279.
 Ahmad, F. and McPhie, P. The debaturation of covalently inhibited swine pepsin. Int. J. Peptide Protein Res. Vol. 12, 1978, 155-163.
 McPhie, P. Kinetic studies on the unfolding and refolding of pepsinogen in urea – the nature of rate limiting step. J. Biol. Chem. Vol. 255, 1980, 4048-4052.
 McPhie, P. The origin of the intermediates detected in the folding of swine pepsinogen. J. Biol. Chem. Vol. 257, 1982, 658-693.
 Wada, A., Saito, Y. and Ohogushi, M. Multiphasic conformation transition of globular proteins under denaturing perturbations. Biopolymers Vol. 22, 1983, 93-99.
 Mateo, P.L. and Privalov, P.L. Pepsinogen denaturation is not a 2-state transition. FEBS Lett. Vol. 123, 1981, 189-192.
 Privalov, P.L., Mateo, P.L.,Khechinashvili, N.N., Stepanov, V.M. and Revina, L.P. Comparative thermodynamic study of pepsinogen and pepsin structure. J. Mol. Biol. Vol. 152, 1981, 445-464.
 Amiza, M. A. and Owusu Apenten, R. K. Urea and heat unfolding of cold-adapted Atlantic cod (Gadus morhua) trypsin and bovine trypsin. J. Sci. Food Agric. Vol. 70, 1996, 1-10.