Biomacromolecules 2002, 3, 9-16
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Articles A Multitechnique Approach in Protein/Surfactant Interaction Study: Physicochemical Aspects of Sodium Dodecyl Sulfate in the Presence of Trypsin in Aqueous Medium Soumen Ghosh* and Asok Banerjee Department of Biophysics, Bose Institute, Centenary Building, P- 1/12, C.I.T. Scheme, VII M, Kankurgachi, Calcutta - 700054, India Received November 30, 2000; Revised Manuscript Received September 1, 2001
Interaction of sodium dodecyl sulfate (SDS) with a globular protein, trypsin, has been physicochemically studied in aqueous medium in detail using tensiometric, conductometric, calorimetric, fluorimetric, viscometric, and circular dichroism techniques. The results indicate that SDS-trypsin aggregates start to form at a surfactant concentration higher than the critical micelle concentration of pure SDS micelle. In contrast, the counterion binding decreases in the presence of trypsin. The free energies and enthalpies of micellization, interfacial adsorption, and entropy of micellization associated with the interaction have also been calculated. The values show that the interaction phenomenon is entropy controlled and endothermic in nature. The increase in viscosity is observed for the system of SDS-trypsin cluster above the critical micelle concentration of SDS micelle only. The aggregation number and interface polarity decrease compared to the values of micelles without protein. Circular dichroism spectra show the high R-helical content and unfolded structure of trypsin in the presence of SDS due to strong electrostatic repulsion leading to a probable “necklace and bead” model in the case of biopolymer-surfactant complexes. Introduction Interaction of proteins with surfactants has been extensively studied for over 50 years.1 By use of different types of physicochemical techniques, interactions between cationic and anionic surfactants with a protein have been extensively investigated in vitro.2-12 These interactions are of importance in the fields of industrial, biological, pharmaceutical, and cosmetic systems. Recently, proteases (peptide-bond cleaving enzymes) are widely used in detergent industry, obviously for its very effective role in cleaning and removing stains, etc. Literature survey reveals that physicochemical studies in this area are rare. In most of these studies, binding of an amphiphile to a single protein has been considered. This “surfactant binding” can lead to the unfolding of proteins and sometimes their denaturation. For denatured proteins, two general cases appear:7 (a) mixtures of anionic surfactants with proteins above the isoelectric point (IEP) and (b) mixtures of anionic surfactants with proteins below the IEP. Below the IEP, the protein can be considered as a “cationic” polymer, the interactions with anionic surfactants are dominated by precipitation phenomena, while above the IEP, the interactions can form stable, fully solubilized complexes which can change drastically the topology and conformation * To whom correspondence may be addressed. E-mail: gsoumen70@ hotmail.com. Present address: Faculty, Center for Surface Science, Department of Chemistry, Jadavpur University, Calcutta 700 032, India.
of the protein molecule in solution. Generally, ionic surfactants bind to proteins and the most widely used anionic surfactant, sodium dodecyl sulfate (SDS), denatures proteins more than do cationic surfactants.1,8-12 Relatively, the interactions of protein with cationic and nonionic surfactants are less studied.13 The globular protein trypsin (EC 3.4.21.4) is a serine protease present as a zymogen (trypsinogen) in the pancreas of all vertebrates. Trypsin which functions in digestion and other essential biological processes has a molecular mass of 23 300 Da and consists of 223 amino acid residues.14 The individual chains are held together by six disulfide bridges. One hundred and one of those amino acid residues (41%), including four of the six disulfide bridges, are identical with corresponding sequence positions of chymotrypsin. A trypsin molecule consists of two domains of nearly equal size, the major constituent of each being a set of six antiparallel strands of polypeptide chain laced together into a β-sheet unit by a network of H-bonds.15 The amino group of the N-terminal Ile of trypsin (originally Ile7 of trypsinogen) forms an ion pair with Asp182, which is important in the conformation of trypsin.14 In this molecule, bound Ca2+ ion became evident during the refinement procedure. Of all the digestive endopeptidases, trypsin has the most pronounced substrate specificity, catalyzing hydrolysis of only Lys and Arg bonds due to the presence of an ionic binding site (Asp189) in the
10.1021/bm005644d CCC: $22.00 © 2002 American Chemical Society Published on Web 12/06/2001
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Biomacromolecules, Vol. 3, No. 1, 2002
active center.14 Trypsin is irreversibly inhibited by many naturally occurring trypsin inhibitors (e.g., soybean trypsin inhibitor).14 Surfactant-protein interactions involve various modes of association favored by dipole-dipole, ion-dipole, or ionion forces. Six possible types of associations on polymersurfactant interactions involving either individual surfactant molecules or surfactant clusters are discussed by Nagarajan and Kalpakci.16 The protein-SDS complex has been described by different models.17,18 Results from small-angle neutron scattering (SANS),19 free boundary electrophoresis,20 viscometry,21 quasi-elastic light scattering,22 NMR,23 and fluorescence study24,25 support the “necklace model” of the complex where micelle-like aggregates are formed along the protein chain. This necklace model of protein-surfactant complexes is similar to the complexes formed between surfactants with polymers20,26 and polyelectrolytes.27 In this paper, we have investigated the interaction between SDS and trypsin using a number of techniques such as tensiometry, conductometry, calorimetry, fluorimetry, viscometry, and circular dichroism to understand the nature of different physicochemical processes and to explore the microenvironment of the protein-surfactant aggregates. Experimental Section Materials and Methods. The anionic surfactant sodium dodecyl sulfate (SDS) used in this study was a product of Sigma, USA. Trypsin from bovine (Bos taurus) pancreas was a liophilized, crystalline powder, a product of Merck, Germany. Trypsin was dissolved in double distilled water, and the pH was unadjusted. The dye safranine T (ST) (Fluka, Switzerland) was crystallized twice from an ethanol-water mixture. Double distilled water of specific conductance, 2-4 µS cm-1 at 303 K was used in all preparations. All measurements were taken at constant temperature in a water bath maintained at 298 ( 0.01 K. Tensiometry. The air/solution surface tensions at various concentrations were measured with a du Nou¨y tensiometer (Kru¨ss, Germany) using a platinum ring by the ring detachment technique. The tensiometer was calibrated against water, and the measured surface tension values were corrected following the procedure of Harkins and Jordan.28 Experimentally, the progressive addition of concentrated surfactant solution with the help of a Hamilton microsyringe to the protein solution in the glass vessel was made at constant temperature, and after proper thorough mixing and equilibration, measurement of surface tension was done. The results are accurate within (0.1 dyn cm-1. The detailed procedure can be found elsewhere.29-31 Conductometry. Conductance measurements were done with a Systronics 304 conductivity meter (India) using a cell of cell constant 1.0 cm-1. The measured conductance values were accurate within (0.5%. With the help of a Hamilton microsyringe, the concentrated surfactant solution was progressively added to protein solution taken in a small beaker and then the conductance values were measured after thorough mixing at constant temperature. The procedure can be found in detail elsewhere.29-31
Ghosh and Banerjee
Calorimetry. The enthalpy of micellization of surfactant solution in the presence of protein was measured with the help of a Tronac 458 Isoperibol titration calorimeter (USA). The instrument was calibrated by measuring the heat of neutralization of hydrochloric acid with sodium hydroxide. The calorimeter can measure a minimum heat change of 1 cal. The detailed procedure is reported elsewhere.32,33 Fluorimetry. The fluorescence emission spectra were measured using a F-3010 fluorescence spectrophotometer, Hitachi (Japan), with a slit width of 1 cm. The probe dye used was ST, whose excitation and emission wavelengths were 520 and 587 nm, respectively. The concentration of ST was kept at the order of 10-5 M. All spectra were measured four times in a constant temperature water bath (accuracy was within (0.1 K) and the mean values were processed for data analysis. Viscometry. The viscosities of protein-surfactant solutions were measured using a Cannon-Fenske capillary viscometer with a flow time of 123 s for water placed in a water bath accurate within (0.2 °C. Circular Dichroism. Far-UV circular dichroism (CD) spectra were measured using a Jasco model J-600 recording spectropolarimeter (Japan) attached with a chiller to control the temperature of the Xe lamp and electronic circuit. The instrument was calibrated with d-10-camphor-sulfonic acid. The light path length was 1 mm in each case. The scan speed was 50 nm/min, and five scans were signal averaged to increase the signal-to-noise ratio. Results and Discussion Critical Micelle Concentration (cmc). In aqueous medium, a pure surfactant can form an aggregate known as a micelle after reaching a concentration called the critical micelle concentration. The dependence of surface tension (γ) of aqueous trypsin at constant concentration with varying [SDS], i.e., (γ - log[SDS]) is presented graphically in Figure 1. The intersection between two straight lines for a particular plot indicates the cmc. The surface tension value decreases with increasing [SDS] and after cmc, γ is almost constant. Such types of interactions between protein and surfactant are also studied earlier.34,35 The specific conductance (κ) measurements at constant trypsin concentration with varying [SDS], i.e. (κ vs [SDS]), is exemplified in Figure 2, which shows two distinct breaks in each plot. The first break points correspond to the critical aggregation concentration (cac) where protein-bound micelles are formed and the second breaks to critical micelle concentration (cmc) where free micelle formation begins. Such types of behavior by conductometric method are also observed in polymer-surfactant interaction.36-38 The cac values of SDS in trypsin solution are almost an order of magnitude lower than the cmc values of SDS in water alone and the cmc of SDS is observed to increase with increasing [trypsin]. The calorimetric titration experiment consists of a series of consecutive additions of concentrated solution of SDS (concentration . cmc) from a buret to the trypsin solution taken in a calorimeter sample vessel. The heat of dilution of SDS in aqueous trypsin solution at a constant concentration
Biomacromolecules, Vol. 3, No. 1, 2002 11
Protein/Surfactant Interaction
Figure 1. Surface tension (γ) vs log [SDS] plots for the mixed systems of SDS-trypsin at 298 K. Effect of trypsin concentration (g dL-1): I, 0.001; II, 0.005; III, 0.01; IV, 0.05. Ordinate scales are indicated on the plots.
Figure 3. Plots of temperature (millivolt) vs time of titration for SDS in the presence of different [trypsin] at 298 K. The crossing point represents cmc. S and E represent the start and end of a run. I, II, III, and IV represent 0.001, 0.005, 0.01, and 0.05 g dL-1 trypsin concentration, respectively. Table 1. Critical Micellar Concentration (cmc) of SDSa and SDS-Trypsin Mixtures in Aqueous Medium by Different Methods at 298 Kb cmc × 103/mol dm-3
b
Figure 2. Specific conductance (κ) vs concentration profile of SDS in the presence of trypsin at 298 K. Effect of trypsin concentration (g dL-1): I, 0.001; II, 0.005; III, 0.01; IV, 0.05. Ordinate scales are indicated on the plots.
is recorded, and typical thermograms are presented in Figure 3. From the flow rate of the buret, the volume of SDS added is calculated at the time corresponding to the crossing point in the figure and then the concentration of SDS at that crossing point is determined by a simple calculation. The crossing points represent the cmc values.32,33 The thermo-
[trypsin]/% w/v
tens
cond
cal
0 0.001 0.005 0.010 0.050
7.94 8.91 10.00 10.60 11.20
7.94 9.80 (1.6) 10.00 (1.7) 10.20 (1.8) 10.40 (2.0)
7.37 7.42 7.84 9.40 10.70
a The cmc values of SDS solution were taken from the refs 30 and 33. The values in the parentheses indicate cac.
grams are endothermic in nature and provide the enthalpy of micellization. It is noticeable that a calorimetric study of a protein-surfactant interaction is very rare.39 The cmc values of SDS alone and in the presence of trypsin solutions at different concentrations obtained from tensiometric, conductometric, and calorimetric methods are presented in Table 1. The experimental results show that with increasing concentration of trypsin, the cmc value of SDS increases indicating strong interaction between SDS and trypsin. Like anionic amphiphiles, DS- of SDS binds with or gets adsorbed on the trypsin molecule and a complex is formed. So the micellization process is hindered and the amphiphile undergoes self-organization resulting in the formation of micelles at a higher concentration than that of pure SDS solution.40 It is observed from Table 1 that at a lower concentration of trypsin, lower cmc values are obtained calorimetrically than values obtained with the other two methods, while the comparable cmc values are obtained by surface tension and conductance methods on the whole. Such method-dependent cmc values have been reported earlier.41
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Ghosh and Banerjee Table 3. Thermodynamic Behaviors of SDSa and SDS-Trypsin Mixtures at 298 K
Figure 4. Experimental cac/cmc ratio vs [trypsin] plot from the conductometric method at 298 K. Table 2. Interfacial Behaviors of SDSa and SDS-Trypsin Mixtures at 298 K [trypsin]/ % w/v
πcmc/ erg cm-2
106Γmaxtot/ mol m-2
Amintot/ nm2
f
0 0.001 0.005 0.010 0.050
44.0 25.0 20.0 26.3 29.0
0.77 0.90 0.93 1.09 1.12
2.16 1.84 1.78 1.52 1.48
0.72 0.48 0.50 0.55 0.57
a
The values of SDS were taken from the ref 30.
In the past, the observation that cac/cmc ) 1 was interpreted as an indication that the polymer and the surfactant do not form complexes.42 The experimental ratio cac/cmc is plotted as a function of [trypsin] in the Figure 4, which shows the increasing tendency of formation of complex with increasing concentration of trypsin. Interfacial Activity. Surfactants orient at the air/water as well as air/protein solution interface and decrease surface tension of water or protein solution. The interfacial adsorption per unit area of surface at various concentrations of surfactant can be calculated with the help of the Gibbs adsorption equation. A detailed explanation of the Gibbs adsorption equation has been reported in the literature.5,30,43 On the basis of surface tension results, the total maximum surface excess, Γmaxtot, at cmc has been evaluated following the modified Gibbs adsorption equation5 Γmaxtot ) 1/2.303RT
lim
CDS-fCDS-(at cmc)
dπ/d log CDS- (1)
and the total area minimum value (Amintot) at cmc is calculated by the equation Amintot ) 1018/NΓmaxtot
(2)
Here, the surface pressure, π ) γwater - γsolution, C represents the surfactant concentration in mol dm-3 and N is Avogadro’s number. The value of (dπ/d log C) is obtained from the slope of the linear plot of π vs log C (plots are not shown to save space). The evaluated values of πcmc, Γmaxtot, and Amintot are presented in Table 2. In the absence of trypsin, the Γmaxtot at cmc corresponds to Γmaxtot in aqueous medium which is minimum and different from those obtained in the presence of trypsin. The interface contains trypsin molecules with DSions. From Table 2, it is observed that Γmaxtot increases with increasing concentration of trypsin and so Amintot decreases. Energetics of Micellization and Interfacial Adsorption. The energy calculations were performed using the equation ∆Gm° ) (1 + f)RT ln cmc
(3)
[trypsin]/ % w/v
-∆Gm°/ kJ mol-1
∆Hm°/ kJ mol-1
∆Sm°/ J mol-1 K-1
-∆Gad°/ kJ mol-1
0 0.001 0.005 0.010 0.050
20.61 17.31 17.11 17.46 17.47
-3.80 0.82 0.91 1.43 3.09
56.41 60.84 60.47 63.39 68.99
77.75 45.08 38.52 41.59 43.35
a
The values of SDS were taken from the ref 30.
for ionic surfactant solution where ∆Gm° represents the standard free energy of micellization per mole of monomer unit and f is the fraction of the counterions bound to the micelle obtained conductometrically described by Ghosh and Moulik.29-31 The value of f is presented in Table 2. The counterion binding for pure SDS solution is maximum, and in the presence of trypsin, f values are lower than that of pure SDS although they show an increasing trend with the increasing concentration of trypsin. The standard free energy of interfacial adsorption (∆Gad°) at the air/saturated monolayer interface is calculated by the relation29-31 ∆Gad° ) ∆Gm° - πcmc/Γmaxtot
(4)
where πcmc is the surface pressure at the cmc. The standard enthalpy of micellization (∆Hm°) can be obtained by the calorimetric method.39,44 The standard entropy of micellization (∆Sm°) has been obtained from the Gibbs-Helmholtz equation ∆Gm° ) ∆Hm° - T∆Sm°
(5)
The values of ∆Gm°, ∆Hm°, ∆Sm°, and ∆Gad° are presented in Table 3. The values of ∆Gm° of SDS-trypsin mixtures are lower than that of pure SDS micelle and are more or less the same. The value of ∆Gad° is the maximum for the SDS solution in the absence of trypsin and decreases in the presence of trypsin. A very low heat of micellization (∆Hm°) is observed in pure SDS solution, and this value increases with increase in concentration of trypsin. For trypsin-SDS interaction, ∆Hm° and ∆Sm°, both are positive. Such positive values for other protein-surfactant systems are also reported in the literature.6 The entropy value (∆Sm°) is higher in the system of SDS-trypsin mixture than that in pure SDS micelle. For the maximum concentration of trypsin (0.05%), the values of ∆Hm° and ∆Sm° are maximum. Such types of interactions are mainly entropy controlled processes. Calorimetric investigations provide information on the thermodynamics of protein denaturation. During the micellization of pure SDS, heat is released (negative value of ∆Hm°) and the process is exothermic in nature. The binding of SDS clusters to the trypsin is essentially athermal, and the initial binding of the surfactant to the protein at low concentration involve SDS monomers. In aqueous media, micellar binding with complete shielding involves some aggregate of the surfactant molecules with absorption of heat (positive value of ∆Hm°) and the entire process is endothermic in nature.38 At higher binding, endothermic contributions arising from
Biomacromolecules, Vol. 3, No. 1, 2002 13
Protein/Surfactant Interaction
Table 4. Aggregation Number,a Dielectric Constant,a and Binding Constant (Kc)b for SDS and SDS-Trypsin Mixtures at 298 K [trypsin]/ % w/v
aggregation no., n
dielectric constant, D
Kc × 10-5
0 0.001 0.005 0.010 0.050
72.0 70.6 68.7 66.4 64.0
29 27 25 23 22
1.27 1.28 1.27 1.25 1.26
a The n and D values for pure amphiphiles were taken from the refs 29-31 and 46. b Kc was obtained according to eq 6, using fluorescence data.
Figure 6. Dielectric constant (D) vs aggregation number (n) profile for pure SDS and mixed combinations of SDS-trypsin according to Table 4.
Figure 5. (1 - FR)-1 vs [S]/FR plots following eq 6 for pure SDS and mixed systems of SDS-trypsin at 298 K: I, SDS; II, III, IV, and V represent 0.001, 0.005, 0.01, and 0.05 g dL-1 trypsin, respectively. Ordinate scales are indicated on the plots.
the unfolding of the protein (supported by our CD spectra) are important in the overall process.1 Aggregation Number and Dielectric Constant. Information on the nature of protein-surfactant aggregates can be obtained from fluorescence probe studies.1 The use of fluorescent probes to obtain information about micellar environments is comparatively new.45 The aggregation number of protein-surfactant aggregate has been estimated by the static fluorescence measurements using the equation46 1/(1 - FR) ) (Kc/n)([S]/FR) - Kc[DT]
(6)
where FR ) (F - F0)/(Fmax - F0). Here, F0 and F are the fluorescence intensities without and with surfactants in protein medium, respectively, and Fmax is the maximum value of F with surfactant in protein medium; S, DT, and Kc represent surfactant, total dye (ST), and dye-micelle binding constant in protein medium, respectively. The values of Kc and n remain invariant at moderate concentration above cmc of surfactant but vary at concentrations considerably above the cmc; n also depends on concentration of surfactant.47-49 The aggregation of protein-surfactant micelles takes place at a surfactant concentration higher than the cmc of micelles without protein. The experimental results of the SDS-trypsin system are graphically presented in the Figure 5 following the equation (6) by plotting 1/(1 - FR) against [S]/FR to determine n and Kc from the slope and the intercept, respectively. The values of n and Kc are presented in Table 4. The dielectric constant of the protein-surfactant complexes has been determined by comparing the Stokes shifts in the fluorescence spectra in the micelle-protein media with that in solvents of known
dielectric constant. The procedure and data analysis are in the literature.29-31,46 The dielectric constant values are presented in Table 4. The values of aggregation number and dielectric constant of SDS are found to decrease with increase in concentration of trypsin to a certain extent. As aggregation begins, the probe passes from an aqueous medium into the more hydrophobic environment and there may be a connection with the distribution of surfactants between aggregates and the free and molecularly adsorbed states and the value n decreases. The fluorescence intensity values of trypsin solution at various concentrations in the absence of SDS are almost the same indicating that no interaction occurs between trypsin and safranine T. But an interaction is involved between safranine T and the protein at the micellar surface. The protein-surfactant aggregates may be exposed to the aqueous surface when aggregation number decreases and the probe molecule is solubilized in the micelle when dielectric constant decreases indicating a less open and less hydrated palisade layer of the aggregated surfactant phase as compared to regular micelles.50 Another explanation for reducing dielectric constant of the micelle may be that the addition of polymer increases the size of micelles which induce a decrease in the area per headgroup with a simultaneous decrease in the interface polarity.51,52 Such a decrease in dielectric constant is also reported in the surfactantpolyelectrolyte system.53 The n and D values of SDS-trypsin mixtures are plotted in Figure 6 and produces a straight line. This n-D correlation is an important physicochemical behavior of protein-surfactant complex that needs further investigation. The probe and surfactant molecules adsorb in specific sites on the protein and probably transferred into the micelle-like aggregates.25 The low values of n suggest that micellar clusters are smaller than free micelles. Viscosity. Viscometry is effective in probing conformational and rheological changes in interaction of protein with ionic surfactant.1 The relative viscosity of surfactant-doped trypsin solution (ηr) vs surfactant concentration, [SDS], is plotted in Figure 7, where the relative viscosity is defined
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Biomacromolecules, Vol. 3, No. 1, 2002
Ghosh and Banerjee
Figure 7. The profile of ηr vs [SDS] (relative to surfactant) for SDStrypsin mixtures at 298 K. Effect of trypsin concentration (g dL-1): (O) 0.001; (4) 0.005; (0) 0.01; (b) 0.05.
as the ratio of the viscosity of the surfactant-doped protein solution to that of the surfactant solution. Two characteristic concentrations in each plot are identified in Figure 7. The first break occurs at [SDS] ) 0.003 mol dm-3, which may be called the threshold concentration, marking the onset of thickening.7 The second break occurs at [SDS] ) 0.015 mol dm-3 and then the relative viscosity curve rises smoothly at a slower rate than the previous one with a higher concentration of SDS. The initial increase in viscosity is due to the charging up of the protein molecule by binding of surfactants.1,54 The subsequent increase of viscosity at high surfactant concentration is possibly due to the cross-linking of the several aggregates by free surfactant micelles in the solution.1 Obviously, with higher concentration of trypsin, the relative viscosity of solution is also higher. The reduced viscosity, ηRed, is expressed as ηRed ) ηsp/C ) (ηr - 1)/C
(7)
where ηsp is the specific viscosity and C is the concentration of protein solution in g/100 mL. The plots of ηRed vs [trypsin] relative to surfactant at different [SDS] are exemplified in Figure 8. None of the plots is linear; the nonlinearity increases with increasing [SDS]. Actually, at higher [SDS], the curve falls off more steeply than the lower one. At a fixed [SDS], amphiphile forms enhanced aggregates in a dilute trypsin solution; the ionic repulsion effect among the attached DS- ions causes the protein coil to expand with increase in reduced viscosity values. The extrapolations of the reduced viscosity curves to zero protein concentration are shown to evaluate intrinsic viscosity values, [η], where [η] ) lim (ηsp/C) Cf0
(8)
Figure 9 shows the effect of SDS concentration on [η] of trypsin-SDS mixtures. The plot shows that the [η] value is high at high [SDS] indicating an expansion of protein coil in the cluster. This conformational expansion is probably a result of enhanced electrostatic repulsion. At this stage, the
Figure 8. Reduced viscosity, ηRed vs [trypsin] (relative to surfactant) for SDS-trypsin mixtures at various concentrations of SDS at 298 K. Effect of SDS concentration: (O) 0 M; (0) 0.003 M; (4) 0.008 M; (3) 0.015; (b) 0.05 M; (2) 0.1 M.
Figure 9. Intrinsic viscosity [η] vs [SDS] for SDS-trypsin mixtures at 298 K.
system can be considered to consist of a necklace-shaped structure55,56 made of trypsin chains loaded with bound micelles. Circular Dichroism. Solutions for CD are prepared by mixing of the trypsin solution in water at different concentrations and 0.1 M SDS solution in equal proportion. The farUV-CD spectra for trypsin in the absence and presence of SDS at 200-250 nm wavelength are shown in Figure 10. The spectrum (B) for pure trypsin differs from the spectra for trypsin-SDS complexes. SDS changes the CD exhibited by trypsin near 210 and 220 nm. The spectra (specially G, H, and I) for complexes show a hump at 220-222 nm at the high concentrations of the trypsin. This indicates the less random coil with the increase in R-helix57 of the trypsin molecule to which SDS binds. These spectra also show a minimum at
