A fluorescence study of the interactions between κ- and αs1-casein and between lysozyme and ovalbumin

Biochimica et Biophysica Acta, 351 (1974) 21-27

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36698 A F L U O R E S C E N C E S T U D Y OF T H E I N T E R A C T I O N S B E T W E E N x- A N D as~CASEIN A N D B E T W E E N LYSOZYME A N D O V A L B U M I N

S. NAKAI and C. M. KASON Department of Food Science, University of British Columbia, Vancouver V6T 1 W5, B.C. (Canada)

(Received November 6th, 1973)

SUMMARY A fluorescence polarization method based on the anisotropy addition law was used for determining the association constants (K) for the interaction between x- and asl-casein in comparison with the lysozyme-ovalbumin interaction. The association constants were 3.2.104 M -1 with a reaction ratio of unity for both K-as~-caseins and lysozyme-ovalbumin in a phosphate buffer, pH 6.8, I = 0.02. Whereas the interaction was most stable at neutral pH for lysozyme-ovalbumin, K for as~-X-caseins increased as the p H decreased down to 6.0. Increases in ionic strength weakened the interactions and the lysozyme-ovalbumin interaction was considerably more sensitive than the a~-x-caseins to ionic strength changes. The association constant for as~x-caseins decreased at lower temperatures, however, the changes were slight for lysozyme-ovalbumin. Amino blockings and decarboxylation, which decreased stabilizing ability of x-casein for calcium a~l-caseinate, decreased K, suggesting that the decrease in stabilizing ability of x-casein was due to weaker interactions with a~l-casein. The interaction between lysozyme and ovalbumin was electrostatic whereas the r - a ~ casein interaction was more hydrophobic in nature.

INTRODUCTION Since the pioneer work of Noble and Waugh [1] a variety of methods have been suggested for studying the interaction of K- and a~l-caseins to elucidate the stabilizing action of r-casein for asl-casein in the presence of Ca 2+. We have been using fluorescence polarization for determining the equilibrium constant of this interaction and the results were reported earlier [2]. However, this method is applicable only to systems for which spontaneous interaction could be expected under certain conditions (for example, at 40 °C for K-asl-caseins [1, 2]). Rawitch and Weber [3] calculated the equilibrium constant of the lysozymethyroglobulin interaction from fluorescence anisotropy and we found that this method is more versatile for studying interactions between proteins. However, the versatility is rather restricted when the levelling-off value of fluorescence polarization (P) is used as the all-bound value (Pb) as in the procedure of Rawitch and Weber [3]. When the association constant (K) is small t h e P vs molar ratios of interacting proteins curve

22 will never level off at experimentally practical ratios. We have modified their method to extend its use to different interactions under various circumstances. Meanwhile, the interaction between lysozyme and ovalbumin has been studied by a frontal gel chromatography [4, 5] and by the sedimentation equilibrium [6].This interaction is of particular interest as it is assumed to be electrostatic [7]. The study of this interacting system will be valuable in order to contrast with the K-asl-casein interaction which was claimed to be hydrophobic in nature [2]. This paper describes the results of our study using an improved fluorescence polarization technique on the K-as~-casein interaction in comparison with the interaction between lysozyme and ovalbumin. MATERIALS AND METHODS K- and asl-caseins were prepared by the method of Zittle and Custer [8] and of McKenzie and Wake [9]. Lysozyme (salt free, 10 900/z/g) was purchased from Worthington Biochemical Corp. and crystallized, salt free ovalbumin (Grade V) was from Sigma Chemical Co. Dansylation of Gl-casein and lysozyme was done according to Rawitch and Weber [3]. Absorbance at 280 nm was used for determination of protein concentration using molar absorptivities of 3.944.105 and 2.97.105 for lysozyme and ovalbumin, and A17oo ~ 2 8 0 n m of 10 and molecular weights of 23 600 and 19 000 for as~- and K-caseins, respectively. Fluorescence polarization was measured as described previously [2]. Stabilization capacity of K-casein was determined by the method of Talbot and Waugh [10]. CALCULATION The procedure of Rawitch and Weber [3] for calculation of K for A + B ~ C was modified as follows: Step 1. K~ values are calculated by substituting an assumed value for Pb, the observed Pi at ratios ([B0]/[A0])i, the ratio of fluorescence yields R, and the polarization of Dns-labelled protein Pf into the following formula: 8, = ( A , -

&)/[(&

-

A,) R + A, -- &]

K~ =/3//(1 --/~) ([Bo] --/~dAo]) A, the fluorescence anisotropy, A = ~ -ff

1), ; fl, the fraction of labelled protein

~

bound; [A0] and [B0], the constituent molar concentrations of proteins A and B; R is calculated as an asymptote K of Y = K + pq"l using six Ri at six ratios of the same intervals [11]. Ri = (I, q- 2 I ± ) d ( I , ÷ 2 I±)f, where I, and I± are the polarized fluorescence intensities at parallel and vertical combinations of polarizers, respectively.

23 Step 2. The coefficient of variation Cv of Kl measured at the different ratios ([B0]/[A0])l, and the regression coefficient b of K~ ----a + b([B0]/[A0])i are calculated. Step 3. Different Pb values are applied in Step 1 for calculation of the average K until the minimum Cv is obtained and b approaches 0. All calculations were programmed in Monroe Calculator 1880. RESULTS

Association constants by fluorescence anisotropy When the procedure for K calculation was applied to an interaction illustrated in Fig. 1 the calculated Ki were scattered for the postulated Pb values of 0.22, 0.23,

0.23 . . . . . . . . . . . . . . . . . . . . . . . . . . (Pb) 0.22

021

/

/

0,20

0.19 0

,b

~

Jo

4'o--%~y-

[So]/i-Ao] Fig. 1. P of Dns-as~-casein as a function of added r-casein amino-blocked with citraconic anhydride. (Pb)

[Ra] × 10-2

Cv (%)

b

0.228 0.230 0.232

7.72 :t: 1.80 6.98 ~: 1.54 6.40 4- 1.42

23.3 22.1 22.3

0.0347 0.0053 -0.0133

0.24 and 0.25 as shown in Fig. 2. A m o n g these, Pb = 0.23 yielded the smallest Cv and b closest to 0. To obtain a more accurate K, the assumed Pb values of 0.228 and 0.232 were used for calculation (Fig. 1). A Pb value of 0.230 was still the best value yielding K of (6.98 ± 1.54)- 102 M -1. The interaction stoichiometry for an asrr-casein interaction in phosphate buffer (pH 6.8, I = 0.02) was determined by fitting the P vs [Bo]/[A0] curve, calculated for different stoichiometries, to the observed P values (Fig. 3). The stoichiometry as~ q- (K)2 ~ C introduced the greatest deviations between the best-fit curve and the observed values. The standard errors of estimate calculated for these deviations were 0.012 for as~ + (r)2 ~- C (Pb = 0.306), 0.0023 for a~ + K ~- C(Pb = 0.303) and 0.0031 for (a~)2 + x ~- C(Pb = 0.306). Thus, the interaction system ofa~l + K ~ C appears to be most suitable for the observed P values. The stoichiometries 2a~ + t¢ ~- C and as~ + 2tc ~ C also did not fit to the observed P.

24

4050

(Pb) = 0.22

(Pb) = 0.23 Cv = 22.1 b = 0.0053

11

!

9 i

y 7 ×

20

5

10 -------•

3

i

I

I

I

I

I

1

I

i

I

(Pb) = 0.25 Cv = 30.1 b = - 0.049

(Pb) : 0.24 Cv = 26.0 b = -0.043

b•

___I

ee

x v

lb 2'0 3'0

o

~o

5b

lb 2'o ~o 2o 5'0

[Bo]/[Ao]

[Bo3/[Ao]

Fig. 2. K vs [Bo]/[Ao] at various levels of assumed Pb.

0.30 /~:.r. ,<'

0.28

,'~z~ :':""

Q28 P 0.24

i¢.

022 0.20 018

dsl * (K) 2 .~- complex 0

d5

I

10

r

15

2'0

25

i

c

i

dsl + K

~

complex

30

~5

b (dsl) 2 + K ~ complex

6

[Bo]/[Ao] Fig. 3. Best-fit curves for interaction between r - and a,l-casein of different stoichiometries. Concentration of Dns-asl-casein, 3.5.10 .5 M in phosphate buffer, p H 6.8, I = 0.02; R = 1.92. - - - - , ast + ( K ) 2 ~ - C ;

--

--,a,,+K~C;

...... ,(c~,)z+K~C.

25 Under the same conditions (pH 6.8, I = 0.02 and 25 °C), similar conclusions were drawn for the lysozyme-ovalbumin interaction. Association constants under various conditions T h e association constants u n d e r various conditions were d e t e r m i n e d by the fluorescence a n i s o t r o p y for the l y s o z y m e - o v a l b u m i n and asl-K-casein systems (Table I). W h e r e a s the l y s o z y m e - o v a l b u m i n in te r a c ti o n was m o s t stable at the neutral p H , TABLE I COMPARISON OF ASSOCIATION CONSTANTS BY FLUORESCENCE POLARIZATION FOR LYSOZYME-OVALBUMIN AND r-a,rCASEIN INTERACTIONS Concentration of labelled lysozyme and asrcasein, 8.10 -6 and 3.5.10 -s M, respectively. Phosphate buffer, pH 6.8, I = 0.02 was used unless specified. Effect of pH pH

Lysozyme-ovalbumin (× 104 M -1)

a~rK-caseins (×104 M -1)

5.0 6.0 6.8 7.8 9.0

1.8 2.5 3.2 3.1 0.85

6.7 3.2 2.5 0.9

I

Lysozyme--ovalbumin

a, rK-caseins

0.02 0.12 0.32

3.2 0.56 0.40

3.2 2.1 1.3

Effect of ionic strength

Effect of temperature °C

Lysozyme-ovalbumin

a~rr-caseins

40 25 15 4

2.8 3.2 1.9 2.5

4.4 3.2 1.6 1.5

Effect of amino and carboxyl blocking asl-K-caseins Control Citraconic anhydride Unblocked Trifluoroacetic acid Carbodiimide

3.2 0.1 0.31 0.02 2.8

Effect of dissociating agents a~rr-caseins 5 M urea 0.005 M sodium dodecylsulfate

0.09 0.03

26 K for asl-x-casein interaction increased as the p H decreased toward 6.0. As the pH of solutions approaches~the isoelectric points, 4.6 and 10.7 for ovalbumin and lysozyme, respectively, the electrostatic interactions should be impaired. Increases in ionic strength resulted in weaker interactions for both systems~and the lysozyme-ovalbumin was appreciably more sensitive to ionic strength changes than the as~-X-caseins. This as well is suggestive of electrostatic interactions between lysozyme and ovalbumin. Similarly, the effect of temperature changes was not the same for the two systems. The association constants decreased more rapidly for the ast-x-caseins than for the lysozyme-ovalbumin as the temperature was lowered, which is indicative of hydrophobic interactions [2]. As expected, urea and sodium dodecylsulfate~markedly diminished the interaction between x- and as~-casein. Amino groups of x-casein were blocked with citraconic anhydride and trifluoroacetic acid as reported previously [12]. The association constant of these deaminated x-caseins for interaction with asl-casein decreased considerably, however, unblocking of amino groups which were blocked with citraconic anhydride partly restored the decreased K. These results account for most of the changes observed in the stabilizing ability of x-casein due to amino blocking as has been reported previously [12]. x-Casein was decarboxylated with a carbodiimide as reported previously [13]. 18 carboxyls of the 20 free carboxyl groups in the x-casein molecule were modified after reaction for 180 min. The extent of esterification of carboxylic acid groups was determined from the increase in the glycine peak using a Phoenix M 6800 amino acid analyzer. As the modified x-casein was only slightly soluble in water at pH lower than 7.0, the interaction study was carried out at pH 7.5. Almost no decrease in K was observed by decarboxylation under these conditions and the stabilizing capacity of this x-casein was 93 ~ of the unmodified when measured at p H 7.5. Thus the decrease in the stabilizing ability of decarboxylated x-casein reported earlier [13] could be due to the solubility decrease of the x-casein caused by isoelectric point shift as a result of decarboxylation. There was no significant difference in K values between the two x-caseins prepared by the methods of Zittle and Custer [8] and of McKenzie and Wake [9]. DISCUSSION In the previous report [2] we used the following relation for calculation of K: P -- PI

F3

Pa - P1

F0

where/5, P1 and Pa correspond to P~, Pf, and Pb in this study. F0 and F3 are the fluorescent intensities of original Dns-asl-casein, and of bound asl-casein, thus F3/Fo = t3, which was used for K calculation [2]. However, Fa/Fo ~ 13 when the fluorescent intensity changes upon interactions. In this case, an approach made by Rawitch and Weber [3] that was derived from the anisotropy addition law (Eqn 6 of Weber [14]) is more versatile for K calculation. Furthermore,/'3 in the previous method [2] was derived from the Perrin plot by extrapolating P of complete interactions. This method

27 o f evaluation o f Pa (Pb in this paper) is not applicable to the interacting systems which are not spontaneous. The m e t h o d proposed in this study enables evaluation o f Pb, and therefore K, even when K is small. The precision of evaluated K was reasonable with a coefficient of variation of 5 ~o which increased as K decreased. M o s t of the mechanical instability o f the fluorescence polarization readings in a spectrophotofluorometer, A m i n c o B o w m a n 4-8202 equipped with a blank subtract photomultiplier 10-267, was obviated by a statistical handling in our calculation procedure as indicated in Fig. 1. As Payens [15] pointed out earlier the stoichiometry of casein interactions is extremely difficult to interpret as caseins self-associate. Similar difficulty was reported for the l y s o z y m e - o v a l b u m i n interaction using a sedimentation equilibrium m e t h o d [6] when the complex was not single. Fluorescence polarization reported in this paper cannot exclude a possibility o f (as1), q- (r), ~- C with r multiplied by n. The changes in K for the asl-r-casein interaction were an almost perfect reflection o f stabilizing ability changes of r-casein. This m a y account for decreased stabilizing ability for asl-casein in the presence o f Ca 2+ induced by chemical modifications o f r-casein. Causes for decreased stabilizing capacity must be located in the mechanism o f interaction between the two caseins rather than in the calcium-binding mechanisms o f ast-casein which m a y be affected by addition o f ,:-casein. REFERENCES I 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Noble, Jr, R. W. and Waugh, D. F. (1965) J. Am. Chem. Soc. 87, 2236-2245 Clarke, R. and Nakai, S. (1971) Biochemistry 10, 3353-3357 Rawitch, A. B. and Weber, G. (1972) J. Biol. Chem. 247, 680-685 Nichol, L. W. and Winzor, D. J. (1964) J. Phys. Chem. 68, 2455-2463 Nichol, L. W., Ogston, A. G. and Winzor, D. J. (1967) Arch. Biochem. Biophys. 121, 517-521 Howlett, G. J. anal Nichol, L. W. (1973) J. Biol. Chem. 248, 619-621 Klotz, I. M. and Walker, F. M. (1948) Arch. Biochem. 18, 319-325 Zittle, C. A. and Custer, J. H. (1963) J. Dairy Sci. 46, 1183-1188 McKenzie, H. A. and Wake, R. G. (1961) Biochim. Biophys. Acta 47, 240-242 Talbot, B. and Waugh, D. F. (1970) Biochemistry 9, 2807-2813 Vann, E. (1972) Fundamentals of Biostatistics, pp. 146-149, Heath and Company, Lexinton, Mass., Toronto and London Nakai, S., Styles, W. G. and Perrin, J. J. (1973) J. Dairy Sci. 56, 690-698 Clarke, R. and Nakai, S. (1972) Biochim. Biophys. Acta 257, 70-75 Weber, G. (1952) Biochem. J. 51, 145-155 Payens, T. A. J. (1966) J. Dairy Sci. 49, 1317-1324