Study of the inhibitory effect of hydrophobic fluorescent markers on the enzymic coagulation of bovine casein micelles: action of TNS

FOOD

HYDROCOLLOIDS ELSEVIER

Food H ydroco lloids 12 ( 1998) 393-400

Study of the inhibitory effect of hydrophobic fluorescent markers on the enzymic coagulation of bovine casein micelles: action of TNS Carlos A. Gatti *, Patricia H. Risso, Stella M. Zerpa Departamento de Qwlnica-Fisica, Facu/tad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario. Suipacha 531 . Rosario 2000, Argentina

Rece ived 20 March 1997; accepted I May 1997

Abstract

The interaction of 2-(p-toluidinyl) naphthalene-6-sulfonate (TNS) with casein micelles (CM) was studied by fluorescence spectroscopy. Fluorescence emission spectra of the complex showed blue shift and intensity enhancement of TNS fluorescence, suggesting the insertion of the marker in low polarity regions of CM. An energy transfer process between the proteins and the marker was detected, showing that most of the TNS binding sites were in the proximity of CM fluorescent residues . TNS inhibited the aggregation step of CM enzymic coagulation, producing probably a decrease of size and amount of aggregates formed. This effect could not be related only to changes in CM net charge, but also possibly to the occupancy of surface hydrophobic regions by the marker. About a 20% decrease in the TNS fluorescence intensity was observed during the proteolytic step of coagulation which could be attributed to release of the marker from its binding sites located in the CM external layer. A bound TNS release was also observed during the initial time of the aggregation step, probably by removal of the bound marker from contact regions between aggregating particles upon their collisions. A further increase, related to the aggregation step, could indicate the uptake of the marker by new hydrophobic sites created in the complex structure of the clusters. The results pointed to the participation of surface hydrophobic regions of renneted CM in their aggregation process. © 1998 Published by Elsevier Science Ltd. All rights reserved .

1. Introduction Casein micelles (CM) are almost spherical casein aggregates stabilized as colloidal suspension in milk. CM are negatively charged, with K-casein located on their surfaces, projecting its hydrophilic moiety to the aqueous environment, thereby providing an important steric component to the particle stability in suspension (Walstra, 1990). Enzymic coagulation of milk starts with a proteolytic step by the cleavage of the K-casein hydrophilic moiety catalyzed by chymosin, followed by a second step of spontaneous aggregation of the resultant paracasein micelles (pCM). Kinetics studies have shown that this aggregation process can be considered as a slow brownian aggregation, following the model proposed by Smoluchowski (Payens, 1979). The presence of relatively high stability coefficient values can be adjudicated not only to electrostatic stabilization because of the remanent negative charge on

• Corresponding author.

the pCM , but also to steric stabilization product of the conformational characteristics of the pCM external layer (Dalgleish, 1983; Gatti , Pires, Orellana, & Pereyra, 1996; Otewwill, 1977; Hunter, 1986; Russel, Saville, & Schowalter, 1991). Therefore, information about surface features of the particles, and possible surface conformational changes upon the partial proteolysis of CM to produce pCM , could be of interest in order to improve our knowledge on the pCM aggregation process, a key process in cheese manufacture. In a previous work , CM interactions with 1- 8 aniline naphthalene sulfonate (ANS) and Nile Red (NR), two hydrophobic fluorescent markers, were studied in order to obtain information on surface hydrophobicity of CM and to follow its changes during enzymic coagulation (Gatti , Risso, & Pires, 1995). Both fluorescent probes interact with less polar regions of CM , producing inhibition of pCM aggregation and pointing to the participation of hydrophobic interactions in this process. However, a loss of hydrophobic sites was observed upon proteolysis of K-casein, a result which could be in opposition with other observations reported on hydrophobicit y

0268-005X j98 j$19.00 © 1998 Published by Elsevier Science Ltd. All rights reserved PII : S0268-005X(98)00037-X

394

C.A. Galli eta!./ Food Hydrocolloids 12 (1998 ) 393--400

of K-casein and para-K-casein (Ono, Yada, Yutani, & Nakai, 1987; Peri, Pagliarini, Iametti, & Bonomi, 1990). A related study was conducted in this work, using 2(p-toluidinyl) naphthalene-6-sulfonate (TNS) as a hydrophobic marker, strongly fluorescent when bound to proteins, in order to confirm and broaden preliminary information on CM and pCM surface hydrophobicity, and its behaviour during the enzymic coagulation process. TNS was chosen for this propose because its fluorescence when bound to proteins is very sensitive to the local environment of the binding sites. This marker exhibits also a high efficiency for energy transfer from the intrinsic protein fluorophors, making it specially useful to follow protein conformational changes (Lin, Eads, & Villafranca, 1991; Morris, Bradley, Campagnoni, & Stoner, 1987).

2. Materials and methods

2.1 . Materials

TNS as potassium salt was purchased from Sigma Chemical Co. and used without further purification. Stock solution nearly 0.5 mM for TNS was prepared in water and stored in the dark at 4°C. The concentration of the stock solution was determined by absorbance measurement, using s = 22 700 M - 1 cm - 1 at 320 nm. Commercial liquid rennet was a gift of C.O.T.A.R. S.A. (Argentine) with a strength of 100 RU, where 1 RU is the activity required to coagulate IOml of substrate, i.e. reconstituted low-heat skim milk powder ("' 10.7% w j w) in 0.01 M calcium chloride at pH (nonadjusted) "'6.35, in I 00 s under the conditions specified in the IDF Standard !lOA: 1987. Absorbance measurements were made on a Beckman DU 640 spectrophotometer. For fluorescence intensity (FI) measurements either a Jasco F P-77 or a Shimadzu RF-S301 PC spectrofluorometer were used. 2.2. Preparation of CM samples

Suspensions of CM were prepared from commercial non fat dried milk by dilution with 5 mM CaC12 , skimming and filtration through glass fiber filters (Whatman GF/A) in order to retain fat globules (Horne, 1986). Protein concentration was determined by the Kuaye method (Kuaye, 1994). Dilutions of the CM suspensions for binding or coagulation studies were prepared in I 0 mM tris- HCI, pH 7.0 for binding or pH 6.4 for coagulation, with I 0 mM CaCh. 2.3 . Size variations of CM

Possible changes of CM size in the dilutions or by action of TNS were followed by the wavelength (A)

dependence of turbidity (<) measured as ex= -d (log -r)/d(log },) (Horne, 1986). r:t. was obtained from the slope of log "! versus log }, plots, in the 400-600 nm range. 2.4. Spectrojluorometric study of the binding

Since high inner filter effect are present even in highly diluted CM suspensions because of turbidity, and quenching and filtering effects also appear at low concentrations of TNS, fluorescence measurements corrected for these effects are possible only in a narrow range of CM and ligand concentrations. These restrictive characteristics of the system make impossible the application of several of the method currently used to analyse fluorescence binding data . Nevertheless, a combination of the methods proposed by Wang and Edelman and Leskovac et a!. enabled us to estimate the values of the apparent association constant (k), and the amount of marker moles bound at saturation per protein mol (n) (Leskovac, Trivic, & Pantelic, 1993; Wang & Edelman, I 971 ). Working at constant CM concentration and varying ligand concentration, and assuming a single type binding site model, Wang and Edelman obtain: I I !iF= !iFM

I

I

+ k!iFM I

(!)

where !iF is the difference between the relative fluorescence intensity of the ligand-CM complex (Fie) and the fluorescence intensity of the so le ligand (FI 0 ), at a given ligand concentration I, and !iFM is the value of !iF when a ll the protein molecules are saturated by the li gand. Eq. (I) results valid when I approach to the free ligand concentration, a condition which tends to be satisfied at high ligand concentrations. Thus, this relation can be used to determine !iFM from the intercept at the origin, by linear regression of the initial part of the curve. The data treatment proposed by Leskovac et al. (I 993), on the other hand, enables us to obtain, for a single type binding site model, the following equation:

(2)

where B = {[(FI/Flo) - 1]1} was calculated for pairs of 1 values (/, and 12), maintaining constant the total CM concentration m; A = (8/ y- I), where 8 is the FI of a I M solution of the ligand- CM complex and y is the FI of a I M solution of the free ligand. This equation enables us to determine n without extrapolation of FI values to infinite ligand concentration.

C.A . Galli et a/. fFood Hydrocolloids 12 ( 1998) 393--400

Finally, t:J.F can be expressed as a function of I applying the following equation:

t:J.FM)

t:.F = ( kmn {I+ k I+ k mn 2

-[(I

+ kl + kmn)

2

-

2

(3) 12

4k lmn] 1

(4)

where Flcorr is the corrected FI value and Flobs the observed one, and Acxc and A cm are the absorbances at excitation and emission wavelength, respectively. When the absorbance at Aem was negligible, Eq. (5) was used for the correction of FI values (Coutinho & Prieto, 1993):

= Ffobs -Ab AJ

(I - 10-AJ) I - IO -Ab

then depend on pCM aggregation kinetics, and accepting the mechanism proposed by von Smoluchowski for colloidal flocculation (Payens, 1979), the concentration decrease of pCM to form doublets will initially follow a second order kinetics:

J

k values were calculated by fitting the experimental data with Eq. (3). The Fie was also measured and fluorescence emission spectra were recorded during CM renneting and aggregation, in order to follow the surface hydrophobicity changes produced upon the processes. Samples for spectra determination or FI measurement were prepared by adding to 2.5 ml of 1/2000 vjv CM dilution (buffer pH 7.0) the required volume of 0.4 mM TNS solution. Fie values were obtained by subtracting the FI of CM dilution from the FI of the mixture. All FI values were corrected applying the following relation (Rendell, 1987):

Ffcorr

395

(5)

where Ab is the absorbance of the CM- TNS complex and Afis the absorbance of the sole ligand, at excitation wavelength. For CM- TNS complexes, 332 and 430 nm were used as excitation and emission wavelengths, respectively. The binding was also studied following the Fie produced by energy transfer from the CM fluorophors, using in this case 281 nm as excitation wavelength, and 345 and 430 nm as emission wavelengths for CM and CM- TNS complexes, respectively. All wavelengths values were determined from fluorescence excitation and emission spectra of the systems under study. 2.5. Coagulation kinetics

Enzymic CM coagulation was studied at high enzyme concentrations, in conditions in which the first proteolytic step can be almost completed in a short time at the start of the process (Carlson, Hill, & Olson, 1987a,b; Gatti & Pires, 1995). The overall rate of coagulation will

2 dN) ( dt o= -kzNo

(6)

where N is the number density of primary particles and k 2 , a second order rate constant, is determined by the diffusion rate of the particles and the efficiency of their collisions. At initial times N"' No (initial number density of pCM). At constant N 0 , the initial rate of decrease of pCM number density, (dNfdt) 0 , can be estimated by the initial rate of increase of the turbidity (dr/dt) 0 , (Dalgleish, 1979; Russel et al., 1991). However, since (dr/dt) 0 depends on the scattering cross section of primary particles and doublets, this parameter results useful for aggregation rates comparison only for samples of primary particles of similar size. The kinetics of CM coagulation in the presence of TNS was studied following the turbidity of the system at 600 nm. Adequate amounts of TNS solutions were added to 2.5 ml samples of 1j100 vfv CM dilution in buffer at pH 6.4, vortexing the mixtures and transferring them to a I em cuvette in a thermostatically controlled, jacketed cuvette holder at 35°C. To start the coagulation, 100 )ll of a 1/ 8 vjv dilution of rennet were added to the cuvette. The mixture was gently stirred with a Teflon stirrer for 5 s, and the absorbance of the micelles suspension at 600 nm was recorded before adding rennet and until a maximum in the absorbance value was reached . The concentration of rennet used was sufficient to hydrolyse casein maximally in a short time at the start of the process, as shown following the release of the caseinomacropeptides (CMP), peptides soluble in 30 g/1 trichloroacetic acid (TCA) by the Lowry- Peterson's method (Peterson, 1977; Queiroz Macedo, Faro, & Pires, 1993). Turbidity was measured as absorbance using rectangular optical glass cuvetts with frosted lateral walls. Turbidity values were plotted as a function of time, and the initial rate of increase of turbidity was graphically calculated as the maximal value of (dr/dt) after the total release of CMP. The Fie was also followed during the renneting and coagulation processes. In these cases, a I/800v/v CM dilution mixed with the desired concentrations of the marker solutions was used. The mixtures were transferred to a fluorescence cuvette placed in the spectrafluorometer into a thermostatically controlled, jacketed cuvette holder at 35°C, and coagulation was started by addition of 12.5 J..ll of 1/8 vjv dilution of rennet. Fl was recorded during the time necessa ry to achieve

C.A. Galli et al. fFood Hydrocolloids 12 ( 1998 ) 393-400

396

agglomeration at all the excitation and emission wavelengths that were used for the study of the binding. The relative variation of fluorescence , !::iF/ !::!.fiJ, where !::iF is the difference between the FI of the TNS- CM complex with added rennet and the FI of a TNS- rennet mixture at the same TNS and rennet concentrations, and !::!.fiJ is the same difference in absence of rennet, was plotted as time function. The kinetics of the coagulation in the conditions used in these cases was followed by turbidity measurements in parallel experiments. No less than 20 fluorescence emission spectra were recorded at different times during the coagulation.

3. Results and discussion

3.1. Binding of TNS to CM

The fluorescence emission spectra of TNS in the presence of CM at the excitation wavelength of the marker (332 nm), showed a strong blue shift and a FI enhancement [Fig. !(A)]. These results pointed to the existence of a TNS- CM interaction, with insertion of the fluorescent probe into low polarity environments of the CM. 0.6 -

A

rf%

I \

0.4 -

0

0

I D I0

t.....

\D \

0

I D

0.2 -

\

0

I 0

\,

o' oD

0.0

oo

300

• .•

..-..•.••

.•·•a

•

00

.• •

oo

450

• 600

A (nm) , .0 -

B

0.8

:

0.6

t..... 0.4 -

0.2

0.0 300

350

400

450

500

f.. (nm)

Fig. 1. Fluorescence emission spectra. (A) TNS 12.73)lM in absence (e) or in presence of CM 0.027 mg j ml ( 0) at },"'" = 332 nm; (B) CM 0.027 mg j ml in abse nce (- ) or in presence of TNS at different con-

centrations, --: 4.32)lM, .... ; 8.57 )lM , - ·- ·- : 12.73)lM, when "-m= 281 nm. Media IOmM Ca 2 + , IOmM tris- HCl buffer system, pH 7.0, T: 26°C.

TNS excitation spectrum overlaps an important area of CM fluorescence emission band, conditions in which high efficiency CM- TNS energy transfer can be expected . Emission spectra obtained using 281 nm as excitation wavelength, showed the presence of an energy transfer process between protein residues and the bound marker evidenced by the quenching of the protein fluorescence (CM FI) simultaneously with bound TNS fluorescence enhancement [Fig. l(B)]. Fig. l(B) showed also the existence of an isoemissive point at 395 nm, thereby suggesting that bound TNS molecules present the same quantum yield of fluorescence over the range ofTNS concentrations used (Lin et al., 1991). No shifts were detected in the protein emission band during the binding, a behaviour that suggests the presence of only one kind of energy donors taking part in the energy transfer process. The critical distance R 0 for this donoracceptor system was measured according to Cantor and Schimmel (Cantor & Schimmel, 1980; Lakowicz, 1986) using the corrected emission spectrum of Trp in water, pH 7.0, as reference to correct the CM emission spectrum and determine the CM relative fluorescence quantum yield. The value obtained, 15 A, suggests that the bound TNS which acts as acceptor of this energy transfer process should be located in hydrophobic regions in the neighbourhood of Trp residues. The binding of TNS to CM was studied from the FI increments (!::iF) obtained in the presence of a constant concentration of CM, for increasing concentrations of TNS, as explained in the Materials and methods section. !::iF values were obtained with excitation at 281 nm and emission both at 345 and 430 nm , and also with excitation at 332 nm and emission at 430 nm. In the first case protein fluorophors were excited, and the bound marker located in the neighbourhood of the protein fluorophors was evidenced, both by protein fluorescence quenching at 345 nm and by bound TNS fluorescence enhancement at 430 nm. With 332 nm as excitation wavelength, all the bound TNS was detected, in despite of its location in relation with the protein fluorophors. Curves of !::iF versus TNS total concentration obtained in all the conditions described , were well fitted by a single site binding model [Fig. 2(A) and (B)], giving binding parameters values which are shown on Table I. These results showed an extensive binding of TNS to CM, with an average binding constant similar or higher than the values observed for the binding of this marker to other proteins (Lin et al., 1991; Morris et al., 1987). Comparison of the n values obtained with excitation either at 281 or 332 nm indicated that most of the bound marker (73%) was located in the neighbourhood of protein fluorophors. Since CM are highly porous structures which involve several kinds of casein molecules, the extensive TNS binding observed could be adjudicated to penetration of CM by the ligand and interaction of this last with casein

C.A . Gatti eta!. / Food Hydrocol/oids 12 ( / 998) 393-400 0

0 .32

A

0

0

0 . 24

u...

397

Table I Apparent associati on co nstant (k) and moles of ligand bound a t sa turation per mol of casein determined by fluore scence intensi ty meas urements (n) , using 23 500d as weigh t average molecular weight for casei ns; at 25°C fo r TN S- C M interactions, in 10 mM Ca 2 + , 10 mM tris- HCI buffer solution , pH 7.0, T: 26°C

0 . 16


n (mol/mol) 0.08

-

281 281 332

0 .00 0

10

15

25

20

30

[TNS) (J.LM)

430 345 430

17 .9 ± 0.9 17 ± 3 23.7 ± 1.5

0.97 ± 0.09 0.40 ± 0.o7 0.50 ± 0.05

Brown, & Farrell, 1993). TNS aggregation during the binding process could also contribute to the extent of the binding.

B

0 .08 0

3.2. State of CM in presence of TNS

0.06 0

u...

0 .04


0 .0 0 0

10

20

30

40

[TN S) (J.LM) Fig. 2. Fluorescence intensity increment of TNS at 430 nm in the presence of CM (6.F) as function s of marker concentration. (A) },exc = 281 nm; (BlJ.cxc = 332 nm. Media I 0 mM Ca 2 + , 10 mM tris- HCI buffer system, pH 7.0, T: 26°C. The curves were plotted using Eq. (3).

molecules located anywhere in the CM structure. Evidences of ligand diffusion into CM were already reported by other authors for different cationic and anionic amphiphilic solutes (Green, 1982). In that case, the existence of different kinds of binding sites could be expected, possibly showing different affinities for the marker, and resulting in complex binding isotherms and a complex behaviour of fluorescence spectra upon binding. However, the experimental results obtained, i.e. the presence of an isoemissive point in the emission spectra [Fig. l(B)], the absence of spectral sh ift of both the CM and the TNS emission bands at increasing TNS concentrations in the medium [Fig. I (B)], and a good fitting of the binding curves by a single type binding site model [Fig. 2(A) and (B)], pointed to the existence of a single kind of binding or adsorption which predominates in the experimental conditions used . In this regard, it is interesting to remark that TNS, an anionic amphiphi le, strongly binds to protein hydrophobic regions, specially if these regions include positively charged residues (Morris et al., 1987). For example, K-casein presents such kind of structure, with an important hydrophobic region (from residues 35 to 68) which involves a Lys + residue at the pH used in this work (Kumosinski,

Comparison of average a values for I/ I 00 (2.650±0.005) and 1/800 CM dilutions (2.8 10 ±0.007) suggested that the CM average diameter was slightly shifted to lower values in the higher dilutions, probably due to a partial dissociation of some of the bigger particles. In the presence of TNS at different concentrations, no variations were detected for a values, showing that the interaction with the fluorescent probe did not introduce any change in CM size.

3.3. Effect of TNS on CM enzymic coagulation kinetics Fig. 3(A) shows several of the curves of r versus time obtained for CM enzymic coagulation kinetics in the presence of different concentrations of TNS a t casein concentration of 0.27 mg/ml. In separate experiments, it was determined that the CMP release was completed in a time of no more than I 0 s in the presence of 27 .33!lM TNS (data not shown). (8r/8t )0 was calculate for each of the curves on Fig. 3(A) and its values were plotted as function of TNS concentration s on Fig. 3(B). This plot shows an inhibitory action of the ma rker on the aggregation step of enzymic coagulation, behaviour similar to that already reported for 1- 8 ani line naphthalene sulfonate (ANS) and Nile Red (NR) (Gatti et al., 1995). Since this inhibitory effect was produced in the same way by both a nionic (ANS, TNS) and uncharged (NR) hydrophobic markers, it appears as not specia lly related to CM charge variations by the marker binding. Coagula ted pCM obtained at different TNS concentrations were centrifuged 5 min at 3500 rpm , conditions in which the pCM clusters obtained in the absence of TNS sediment completely. Increasing Fl (Aexc = 28 1 nm , ),em = 430 nm) and increasing turbidity were observed in the supernata nts [Fig. 4(A) a nd (B)], respectively). Similar results were obtained for Fl when 332 nm was used as the excitation wavelength (not shown). These results probably showed the presence of progressively

C.A. Gatti et al. fFood Hy drocol/oids 12 ( 1998 ) 393--400

398 0 . 375 -

A

A

0.15 -

0.360 0 . 10 0 . 3-4!i

0.330 0 .05 -

0 .3 15 0 .00

0.300 -

0

8

0

0 . 04 -

0.12

0 .03

B

0 .09

2

~

"'(]

"1-

________ ,_______- !

B

£ ~

12

(TNS] (fi.M)

(min)

0 . 02 -

1-

0 .0 6

0

"'(]

~

0 0.01

0.03

0 Q

0.00

0 .00 8

12

16

20

24

28

0

12

[TNS] (fi.M) Fig. 3. (A) Turbidity (r) values as function of time for enzymic CM coagu lation in the presence of different concentrations of TNS: e : without added TNS, 0: 4.32 J.1M, V': 8.57J.1M, 0 : 12.73J.1M, t. : 20.82 J.IM, <>: 27.33 J.1M ; (B) (dr/dt)0 values as function of added TNS concentrations. Casein concentration 0.27 mg/ml, 10 mM Ca 2 + , 10 mM tris- HCl buffer system, pH: 6.4, rennet: 100 J.il of 1/8 vfv dilution , T: 35°C. Each experimental point is the average ( ± S.E.) of at least three meas urements.

Fig. 4. (A) FI at 430 nm with excitation wavelength at 281 nm and (B) rat 400 nm of both TN S- CM mixtures at 20 min after the addition of rennet (e), and the supernatants obtained by centrifugation at 3500 rpm 10 min of these mixtures (0), in the presence of different concentrations of TNS. Casein concentration 0.034mg/ml , lOmM Ca 2 + , lOmM tris- HCl buffer system, pH : 6.4, rennet: 12.5J.il of 1/ 8 vfv dilution, T: 35°C. Each experimental point is the average ( ± S.E.) of at least three measurements.

smaller aggregates and even non-coagulated pCM as the TNS concentration increase. The decreasing maximal turbidity reached in the coagulation curves for increasing TNS concentration [Fig. 3(A)] could then be attributed to a shift of the size distribution curve of the aggregates to lower sizes by action of the marker. Therefore, and taking into account that the inhibitory effect increased with increasing binding of the marker, the inhibition could be attributed either to the occupation of hydrophobic regions of the pCM surface, disabling this region to take part in hydrophobic interactions between micelles leading to aggregation, to structural modifications introduced by the marker in the pCM outer layer leading to increased steric stability of the particles, or both. FI were followed during coagulation for a casein concentration of 0.034 mgj ml. FI data were obtained either with excitation at 281 or 332 nm. Both 345 and 430 nm were used as emission wavelengths in the first case, while 430 nm was used in the second case. The time to reach 100% CMP release at the low casein concentration used for these studies was difficult to

measure directly. It was then calculated from kinetic data obtained at higher casein and enzyme concentrations, applying Michaelis- Menten kinetics, and was found to be no longer than 2 min 30 s for 27.33!lM TNS. TNS FI in the presence of rennet alone was negligible, showing that the Fie variations observed can be attributed to TNS- CM interactions. During the coagulation process, the Fie measured at 12. 73!lM TNS obtained by energy transfer, with 281 nm as excitation wavelength (Fig. 5), decreased for a period longer than the time estimated for 100% CMP release reaching a minimum which was about 80% of its initial value. A similar behaviour was observed for the Fie with 332nm as excitation wavelength. Conversely, CM FI measured at 345 nm showed an increase during the same period. After a time of about 20 min, all the measured FI reversed their behaviour, showing increases at both excitation wavelengths, tending to a maximum , while CM FI decreased tending to a minimum . Similar results were obtained using other TNS concentrations (not shown).

399

C.A . Gatti et a/.fFood Hydro colloids 12 ( 1998) 393-400 A

0 .09 0 . 15

'Y" '''' " •··~

......... . ... .,·

•·.. ...

__

;.:·········•

0 .06

0.12

G:

0.09 -

...

0 . 07

0.06 . . .. · · · · ········· {i . . ..... !t .

0 .06

···· ···•

0.03

20

40

60

t

80

1 00

1 20

1 40

(min)

(min)

Fig. 5. Protein fluorescence (CM Fi) and Fi of TNS- CM complexes (Fie) during coagulation. e: CM Fi at 345 nm with excitation at 281 nm, T: Fie at 430nm with excitation at 281 nm, •: Fie at 430nm with excitation at 332nm. TNS concentration 12 . 73~-LM , casein concentration 0.034mg/ml, lOmM Ca 2 +, lOmM tris- HCl buffer system, pH: 6.4, rennet: 12.5~-Ll of 1/8 vfv dilution, T : 35°C.

B 1.05 D

0

LL..

~

Emission spectra (not shown) periodically recorded at both excitation wavelengths, for all the TNS concentrations used, showed only FI changes, with no shifts for any of their emission peaks. Examination of this set of fluorescence data showed, in a first place, that the changes in Fie were related to the chymosin action, which probably affects that fraction of the marker bound in the neighbourhood of the site of the enzyme action in surface K-casein. Fie measured using excitation either at 332 or 281 nm showed similar behaviour, suggesting that chymosin action affects TNS bound near a Trp residue. In fact, in the structure of surface K-casein, Trp 76 could be located near to the Phe-Met (105- 106) bond which is hydrolysed by chymosin (Kumosinski et al., 1993). In the second place, the FI variations observed until 20 min, with a decrease of Fie at both excitation wavelengths used simultaneously with an increase of CM FI, suggested a decrease in the energy transfer process which could be explained by a release of bound TNS because of the cleavage of CMP in surface-K-casein. An inverse process appears at longer times, with Fie increase and CM FI decrease, behaviour which could be explained as a binding of TNS to new hydrophobic binding sites, which appear as a consequence of the pCM aggregation and the formation of clusters of increasing size and complex structure. The simultaneous decrease of CM FI showed that these new binding sites are located near enough to the pCM ftuorophors to permit energy transfer between them and bound TNS. Fluorescence data obtained (A.exc = 281 nm, Aem = 430 nm) at different TNS concentrations and expressed as t:::.Fj t:::.Fl, showed that the increasing inhibition of the aggregation process produced by increasing TNS concentrations resulted also in progressively lower increases of Fie during the aggregation step [Fig. 6(B)].

0.90 -

LL..


0 . 75

0

5

10

15

20

25

JO

(min) Fig. 6. rand relative increment of fluore scence (D.F/ D.f'l) versus time, during coagulation. (A) rat 430 nm; (B) D.F/ D.f'J with excitation wavelength at 281 nm and emission wavelength at 430 nm. TNS concentrations: e : without added TNS, 0 : 4 . 32~-LM , \7 : 8 . 57~-LM and 0 : 2 12.73~-LM . Casein concentration 0.034mgfml , 10 mM Ca +, lOmM trisHCI buffer system, pH : 6.4, rennet: 12.5111 of 1/8 vfv dilution, T: 35°C. Each experimental point is the average of at least three measurements.

Comparison of !:::.F/ t:::.Fl and r measurements in parallel experiments [Fig. 6(A) and (B)] showed that the decrease in !:::.F/ t:::.Fl occurred for a time longer than the lag time between initiation of coagulation (time zero) and the increase in turbidity seen in Fig. 6(A). This fact suggests that the decrease of Fie continued after the primary step of enzymic coagulation was completed . If, as proposed, this decrease was produced by the release of bound TNS, then this release continued during pCM aggregation, probably by removal of the bound marker from the contact region between aggregating pCM, due to collisions between them .

4. Conclusions

The results of this and previous work have shown that fluorescent hydrophobic markers, either anionic or non charged, bind strongly to apolar regions of CM (Gatti et al., 1995; Gatti, Risso, Pires, & Alvarez, 1989). In the case of TNS, the presence of energy transfer

400

C.A. Galli et al.j Food Hydro colloids 12 ( 1998) 393-400

processes suggests also that most of the bound marker is located in the proximity of fluorescent CM residues. In this regard it is interesting to note that recent structural studies of caseins have shown that these proteins could present regions that fulfil most of the characteristics favourable to TNS binding, for example the hydrophobic region from residues 35 to 68 ofK-casein (Farrell, Brown, & Kumosinski, 1993; K umosinski et al., 1993). The release of about 20% of the bound TNS by chymosin action showed that an important fraction of the ligand could be bound in the CM external layer. In fact, pCM external layer involves hydrophobic sites able to bind TNS , i.e. the mentioned K-casein region or other similar casein structures exposed to the medium and accessible to the ligand . If these hydrophobic regions take part in the formation of intermicellar hydrophobic bonds in the lasting contacts between pCM, their increasing occupation by TNS could lead to the impairment of such bonds, hence to the decrease of the number of effective collisions between the particles and to the decrease of their aggregation rate. Moreover, a release of bound marker during the aggregation process, as the analysis of FI data seems to show, could be indicating that bound TNS has to be di splaced from surface hydrophobic sites for the pCM association to occur, suggesting in this way that these sites participate in the linking between pCM . Although previous results with Nile Red , an apolar marker, showed a similar inhibitory effect, the possible contribution of an increase in pCM negative charge due to TNS binding to the diminution of the aggregation rate in this case, should not be neglected.

Acknowledgements

This work was supported by grants from the National University of Rosario (Argentine).

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