The effect of succinylation and acylation on the physicochemical properties of αs1-casein B

Biochimica et Biophysica Acta, 328 (1973) 433-447

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA

36567

T H E E F F E C T OF SUCCINYLATION AND ACYLATION ON T H E P H Y S I C O C H E M I C A L P R O P E R T I E S OF asl-CASEIN B

L. IRONS', M. T. A. EVANS, M. JONES AND P. J. MILLER Unilever Research Laboratory, Colworth/Welwyn, The Frythe, Welwyn, Hertfordshire (Great Britain)

(Received June 29th, 1973)

SUMMARY I. The effect of succinylation and acylation on the association and conformation of asl-casein B has been studied b y ultracentrifugation, optical rotatory dispersion (ORD) and nuclear magnetic resonance (NMR) spectroscopy. 2. Succinylation prevented the association of ast-casein B due to the additional negative charge introduced. 3. Acetyl asl-casein formed an associating system similar to that of the unmodified protein, n-Hexanoyl, n-octanoyl and n-decanoyl derivatives associated strongly with increasing protein concentration and remained in an aggregated form in dilute solution. Propionyl and n-butyryl asl-caseins behaved in a fashion intermediate between the acetyl and n-hexanoyl derivatives. 4. The size of the aggregates produced by the acyl derivatives formed a series similar to that found with corresponding derivatives of fl-casein A, with a m a x i m u m at the n-hexanoyl derivative. This was attributed to enhanced hydrophobic bonding with increasing n-alkyl chain length and to structural differences in the organisation of the associating units. 5. ORD studies showed small differences in secondary structure between derivatives. NMR spectra of derivatives containing long n-alkyl chains revealed sidechain interactions which were not present in the native molecule.

INTRODUCTION

Bovine asl-casein is a phosphoprotein comprising about 45% of the casein component of milk. Four genetic variants are known, asl-caseins A, B, C and D, the B form being the most common. The association of asl-casein B has been studied in some detail by Schmidt and coworkers 1-~. Their work shows that asl-casein B is an example of a rapidly re-equilibrating, associating protein system. The molecular weight of the associating subunit is 23 000-24 ooo (ref. 2) and the association depends more on " Present address: Microbiological Research Establishment, Porton Down, Salisbury, Wilts, Great Britain.

434

L. IRons et al.

ionic strength than temperature 5. Conformational studies show that asl-casein B has little helical structure 8-~° but is still more compact than a completely denatured protein s . This paper describes the effect of acylation and succinylation on the physicochemical properties of as~-casein B, as studied by ultracentrifigation, optical rotatory dispersion and nuclear magnetic resonance spectroscopy. The results are related to the modification of the charge-hydrophobic balance in a protein where both electrostaticS, 3 and hydrophobic forces 5 are important in determining association behaviour. This study is a sequel to previous work on the chemical modification of fl-casein A (reL I I , i2), a protein in which hydrophobic bonding is the principal agent in the association process. MATERIALS

Purified asl-casein B was prepared by the method of Thompson and Kiddy 13. Derivative preparation using succinic and straight chain acyl anhydrides has been described previouslyn,12,14. Purification of the asx-casein B derivatives was carried out by chromatography on DEAE-cellulose in 3.3 M urea, o.oi M imidazole-HC1 buffer, p H 7.0. The succinyl derivative was eluted in a linear O.l-O.6 M NaC1 gradient. The gradient for the n-hexanoyl, n-octanoyl and n-decanoyl derivatives was O.l-O.8 M NaC1 and other asl-casein B derivatives were purified in a O.l-O.45 M NaC1 gradient. All chemicals were of reagent grade quality. Guanidine hydrochloride was prepared from A.R. grade guanidine carbonate 15. METHODS

Starch gel electrophoresis was carried out as described previously n. The substitution of lysine residues was determined by a ninhydrin method TM, while modification of serine and threonine was estimated by an alkaline hydroxylamine procedurelL Protein concentrations were determined by a semi-micro Kjeldahl method using a nitrogen to protein conversion factor of 6.52.

Sedimentation coedficients Sedimentation coefficients were measured in a Beckman Model E analytical ultracentrifuge using schlieren optics. Proteins were dissolved in phosphate buffer 11, p H 7.0, ionic strength o.I and were dialysed against the buffer overnight at 4 °C. The dialysed solutions were allowed to equilibrate at 20 °C for 4 h before sedimentation velocity or molecular weight experiments at room temperature (19-22 °C) were started. Some experiments with asl-casein B were carried out at low temperature (2.0-3.0 °C) immediately following overnight dialysis. Sedimentation coefficients were calculated from the movement of the m a x i m u m ordinate of the schlieren curve and were corrected to standard conditions (s°2o,w). Protein concentrations were corrected for radial dilution during centrifugation. Molecular weights Molecular weights were measured in the ultracentrifuge b y the Klainer and Kegeles is version of the approach to equilibrium method 10 using schlieren optics,

MODIFIED asl-CASEIN B DERIVATIVES

435

Rotor speeds were chosen to give a linear extrapolation of the gradient curve to the meniscus 2°. Apparent weight-average molecular weights (Mw)app were calculated from the air/solution meniscus patterns obtained from ten times enlargements of the photographic plates. Over the time scale of the experiments (about 3 h), no dependence of (Mw)app on the time of centrifiugation was observed.

Partial specific volumes The partial specific volume of as~-casein B at 25 °C was calculated to be o.724 ml/g from the amino acid composition 2~. The partial specific volume at 2 °C was calculated from the value at 25 °C assuming a temperature coefficient of 3.65" lO -4 ml/g per degree 2~. The specific volumes of succinylated and acylated lysine residues were calculated from the molal volumes of the groups by the method of Cohn and Edsall ~a. Assuming modification of all the lysines, the following partial specific volumes were calculated at 25 °C: acetyl asl-casein, 0.725 ml/g; propionyl asl-casein, 0.728 ml/g; n-butyryl asl-casein, 0.732 ml/g; n-hexanoyl asl-casein, 0.738 ml/g; n-octanoyl aslcasein, 0.745 ml/g; n-decanoyl asl-casein, o.751 ml/g; succinyl as~-casein, 0.720 ml/g. If modification of the hydroxyl groups of serine and threonine is also taken into account (see Results and Discussion), the partial specific volumes of the acetyl and n-hexanoyl derivatives become 0.724 and 0.746 ml/g, respectively. However, use of these figures in the calculations of sedimentation coefficients and molecular weights makes less than 1% difference to the values obtained.

optical rotatory dispersion ORD measurements were made over the wavelength ranges 578-313 nm and 250-200 nm with a Polarmatic 62 automatic recording spectropolarimeter (Bendix Electronics Ltd) as previously described11, TM. The mean residue weight of asl-casein B and its derivatives was taken to be 119. 5. The results obtained in the 578-313-nm range were analysed by the Drude, Moffit-Yang and 2-term Drude equations, aHelix content was estimated by the equations H b 0 = - - b o / 6 . 3 o, H225 =

--(Azz~ + 6o)/19.9 a n d H193 = AI~ 3 + 750/36.5

(refs 24, 25).

Nuclear magnetic resonance 22o MHz/s NMR spectra were obtained at 25 °C as single scans on a Varian H R 220 instrument. Protein solutions (0.5-3%) were in ~H~O-phosphate buffer, pH 7.0, 2H20-phosphate buffer-8 ME~H4]urea or trifluoro-E2Hlacetic acid. Resonance positions are given on the z scale. R E S U L T S AND DISCUSSION

Succinylation and acylation of asl-casein B modified over 8o % of the lysine eamino groups. In the acetyl and n-hexanoyl derivatives, all available serine and threonine groups were substituted (Table I). Derivatives purified by DEAE-cellulose chromatography eluted as single peaks and the recovery was good. Starch gel electrophoresis showed that succinyl asl-casein moved as a single band with a mobility about

436

L. IRONS et al.

TABLE I ]~XTENT OF MODIFICATION OF LYSINE AND HYDROXYL GROUPS IN SUCCINYL AND ACYL DERIVATIVES OF asl-CASEIN B

A c e t y l a81-casein P r o p i o n y l asl-casein n - B u t y r y l asl-Casein n - H e x a n o y l asl-casein n - O c t a n o y l asl-casein n-Decanoyl asrCasein Succinyl asl-casein

Lysine groups modified (%)

OH groups modified(%)

92 ioo 95 97 83 82 89

IOO not determined not determined IOO not determined 25 9

the same as that of the unmodified protein. All other derivatives gave rather streaked patterns, extending from the origin to about two-thirds the length of the gel. This

Fig. I. S e d i m e n t a t i o n p a t t e r n s of asl-casein B a n d its s u c c i n y l a n d acyl derivatives. I n p h o s p h a t e buffer, p H 7. (A) asl-easein B, 1 . 1 5 % , a t 20.0 °C, 5o 74 ° r e v . ] m i n , a f t e r 172 rain. (B) aBl-casein t3, o.i2%, at 20. 5 °C, 50 74 ° r e v . / m i n , after 177 min. {C) Acetyl asl-casein, o.67%, a t 2o.o °C, 5 ° 74 ° r e v . / m i n , a f t e r 146 rain. (D) P r o p i o n y l a s l - e a s e i n , 0.50%, at I9.I °C, 59 78orev./rain, after 6o min. (E) n - O c t a n o y l asFeasein, o.48% (top), 0.35% (bottom), a t 20. 5 °C, 59 78o rev./min, a f t e r 45 mira (F) Suecinyl a~l-casein, 1.o9% (top}, o.8o% (bottom), at i9.6 °C, 59 78o r e v . / m i n , a f t e r IO8 rain.

437

MODIFIED asl-CASEIN B DERIVATIVES

shows that even in strong urea, these derivatives are polydisperse with respect to molecular charge or size and, unlike the native protein, are not completely dissociated into subunit forms. The patterns of the n-hexanoyl, n-octanoyl and n-decanoyl derivatives in particular, resemble those of the corresponding/5-casein A derivatives 12. Sedimentation studies

Some sedimentation patterns of a81-casein B and its succinyl and acyl derivatives are shown in Fig. I. The sedimentation pattern of asl-casein B showed a single, symmetrical peak at high protein concentration (Fig. IA), while at low concentration the peak had a distinct trailing edge (Fig. IB). The sedimentation coefficient of asFcasein B initially increased with increasing protein concentration, reaching a maximum between 0.25-0.35% protein concentration, then decresing at higher protein concentrations (Fig. 2). Sedimentation coefficients measured at low temperature were slightly higher than those obtained at room temperature. 4.5

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Molecular weight values obtained for usl-casein B increased with increasing protein concentration and values obtained at low temperature were again slightly higher than those measured at room temperature (Fig. 3)- No maxima could be found in the curves of (Mw)app versus concentration over the range o.1-1.5% (Fig. 3)- For asl-casein B, this dependence of s~0,w and (Mw)app on protein concentration is typical of a rapidly re-equilibrating associating protein system~% Although the association of asl-casein B has not previously been studied in phosphate buffer, pH 7, ionic strength o.I, our molecular weights measured at concentrations above about o.4% are very close to those reported by Ho and Chen 8 in pH 7, KC1, ionic strength o.I. Our results also confirm their observation that aggregates of asl-casein B dissociate slightly as the temperature is raised.

438

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It is well known that both as1- and/~-caseins associate strongly in solution and it has been suggested that hydrophobic bonding 5 is important in the association of these proteins. However, the association of asl-casein B has been found to depend strongly on p H 27 and ionic strength2, 3, but not on temperature 5, which indicates the importance of both electrostatic and hydrophobie interactions in the association. Recently, Schmidt2, 3 has employed light scattering to characterise the association of asl-casein B in p H 6.6 imidazole buffers of differing ionic strengths. The association is best described in terms of consecutive association steps and at an ionic strength of 0.05, it is found that aggregates up to the tetramer are present ~. It has also been shown that at low protein concentrations, further dissociation of the asl-casein B aggregates can occur and at infinite dilution monomers of mol. wt 23 00o-24 ooo are obtained e. Consequently, our S2o,w and (Mw)app versus concentration curves should extrapolate to values of about 2.0 S (ref. 28) and 23 0o0-24 ooo at infinite dilution. However, the schlieren optical system of the ultracentrifuge does not allow us to make measurements on the very dilute protein solutions which are required for this extrapolation. The sedimentation pattern of acetyl asl-casein showed a single peak with marked a s y m m e t r y on the trailing edge (Fig. IC). Prolonged ultracentrifugation did not resolve this trailing edge into a separate peak and the general features of the pattern did not change with increasing protein concentration. Both sedimentation coefficient and molecular weight values for acetyl as~-casein first increased with increasing protein concentration, reached a m a x i m u m and then decreased as the protein concentration increased further (Figs 2 and 3). Over the whole concentration range studied, the sedimentation coefficient and molecular weight values obtained for acetyl asl-casein were much smaller than those of the unmodified protein. It therefore appears that acetyl asl-casein forms an associating system similar to that of aslcasein B but the lower molecular weight values can be explained b y the increased net negative charge of the derivative at p H 7 causing some dissociation of the aggregates.

439

MODIFIED Ctsl-CASEIN B DERIVATIVES

This is analogous to the dissociation of ast-casein B into its monomer form at alkaline pH ~7. The maximum in the (Mw)app v e r s u s concentration curve for acetyl estcasein (Fig. 3) shows that non-ideality effects due to the increased negative charge are important. At low concentrations, both the sz0,w and (Mw)app v e r s u s concentration curves (Figs 2 and 3) may extrapolate to values corresponding to those of the monomeric form of asl-casein B. The sedimentation patterns of propionyl asl-casein (Fig. ID) and n-butyryl aslcasein showed a fairly symmetrical peak at all protein concentrations. At concentration down to 0 . 0 5 0 , no dissociation of these proteins could be detected and there was a linear dependence of the sedimentation coefficient of both proteins and of the molecular weight of the propionyl derivative with protein concentration (Figs 3 and 4). Least squares analysis of the sedimentation results for propionyl asl-casein showed that they could be fitted by the equation: Szo,w

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octanoyl and n-decanoyl asx-caseins. In phosphate buffer, p H 7, at 19-22 °C. O, propionyl aslcasein; O, n-butyryl ast-casein; &, n-hexanoyl asx-casein; ~7, n-octanoyl ast-casein; A, n-decanoyl asx-casein. The points marked 2, 3 and 4 for the n-hexanoyl derivative were obtained from experiments on solutions prepared b y diluting solution i.

440

L. IRONS et al.

these proteins because of their obvious polydisperse nature on sedimentation. For these derivatives, sedimentation coefficients increased with increasing protein concentration and no maxima were found in the S2o,w versus concentration curves. At concentrations down to 0.05%, there was no evidence of dissociation of the aggregates into monomers of mol. wt 23 000-24 ooo and for the n-hexanoyl derivatives tile association was fully reversible with changes in protein concentration (Fig. 4). These derivatives, therefore, associate strongly with increasing protein concentration. The association must take place by the aggregation of assemblies of monomer subunits characterised by S~o,w values of about 6.5 S, 5.8 S and 3-7 S for the n-hexanoyl, noctanoyl and n-decanoyl derivatives, respectively. These values correspond to molecular weights of about I4o ooo, 118 ooo and 60 ooo, respectively28. In contrast to the above results, succinyl ast-casein sedimented as a symmetrical, slow moving peak (Fig. IF), with a linear dependence of I/S2o,w and I/(Mw)app on protein concentration (Fig. 5). Least squares analysis showed these results could be fitted by the equations: - S2o,w

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(3)

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MODIFIED asl-CASEIN B DERIVATIVES

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the fact that we used a buffer of relatively low ionic strength, the effect of non-ideality in our experiments was considerable, thus leading to a large extrapolation of I/(Mw) app to infinite dilution. At pH 7, the net negative charge of succinyl ast-casein is about twice that of the unmodified protein and this increase in charge causes the aggregates normally present to dissociate into the monomer form. A similar effect has been found with carboxyacyl derivatives of fl-casein A (ref. II). Despite this difference in charge, succinyl ast-casein had about the same mobility as as~-casein B in starch gels containing 7 M urea, which indicates that the succinylated protein is in a more expanded configuration. This must be due to increased electrostatic repulsions between the charged residues on the modified protein. The association behaviour of the acylated proteins can be qualitatively compared by their s~o,w values. If we assume that changes in sedimentation coefficients can be largely attributed to changes in aggregate size, then for the acylated aslcaseins, which all have nearly the same net negative charge, we have from Figs 2-4 the series: acetyl ~ propionyl < n-butyryl < n-hexanoyl > n-octanoyl > n-decanoyl. This series is almost identical to the one obtained previously for acyl derivatives of fl-casein A (ref. 12), which suggests a common mechanism of formation of the different kinds of aggregate. The decrease in the aggregate size of asFcasein B on succinylation, acetylation and propionylation can be explained by a simple electrostatic charge effect. The increase in the aggregate size of the acylated asl-casein derivatives from the acetyl to the n-hexanoyl can be explained by assuming that the greater hydrophobic bonding of the longer alkyl chains enhances aggregation. In the same way as for the fl-casein A derivatives 12, we suggest that the decrease in aggregate size from the n-hexanoyl to the n-decanoyl derivative indicates that the associating units of these proteins, which apparently consist of several 23 ooo-24 ooo molecular weight subunits, have different configurations in solution. With such highly hydrophobic derivatives, the molecules within the associating units are probably arranged so that contact between their hydrophobic residues and the solvent is minimised. The intermolecular associations between these units would then depend on the location and accessibility of the hydrophobic groups and the net charge on the protein. The strong, intermolecular hydrophobic bonding between molecules comprising the associating units explains why these proteins do not dissociate into subunit forms on dilution or in starch gels containing 7 M urea. The association of acetyl asl-casein may be similar to that of the unmodified protein and be governed by a combination of electrostatic and hydrophobic effects, unlike the longer n-alkyl chain derivatives, where hydrophobic effects predominate. The propionyl and n-butyryl derivatives appear to gave properties intermediate between those of the acetyl and the n-hexanoyl derivatives. Presumably with these derivatives, the hydrophobic effect of the substituent alkyl chains just balances the electrostatic effects of increased net negative charge.

ORD and N M R The sedimentation studies have shown that marked changes occur in the association of asl-casein B on acylation and succinylation. To determine whether alterations in structure accompanied these modifications, we also carried out ORD and NMR studies.

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The ORD parameters and the far-ultraviolet rotatory dispersion curves of a s l casein B and its succinyl and acyl derivatives are shown in Table I I and Fig. 6. For asl-casein B, our ORD parameters are in reasonable agreement with those of Herskovits 7 and Herskovits and MescantP but they differ slightly from those found by Ho and Chen s and Ikezawa and NiimP. I t is evident from Table II, that the ORD parameters of asl-casein B undergo small changes when the protein is chemically modified or treated with denaturing solvents. The presence of a Cotton effect trough near 233 nm (Fig. 6A) and the agreement in the calculations of a-helix content from the A193 and A~25 parameters of the 2-term Drude equation, suggests that what little secondary structure is present in gs~-casein B, is in the form of an s-helix. Proteins without intrachain disulphide bonds exists in a completely random coil form in 6 M guanidine hydrochloride a3. In 3-6 M guanidine hydrochloride, the ORD parameters of asycasein B remain unchanged (Table II) and are close to those found for the protein in 8 M urea 7. Under these conditions there is a marked decrease in a-helix content. In I - 2 M guanidine hydrochloride, some secondary structure is apparently retained. From Table I I and Fig. 6A it appears that asycasein B loses about half its secondary structure on succinylation. This is a consequence of the disrupti~ e effect of the increase in net negative charge. A similar effect has been found for the carboxyacyl derivatives of/5-casein A (ref. II). Acetyl ast-casein has ORD parameters closely resembling those of the succinyl derivative (Table II), showing that the principal influence of acetylation on the structure of gst-casein B lies in the increase in net negative charge. In contrast both propionyl and n-butyryl asl-caseins have secondary structures which must be almost identical with that of the unmodified protein (Table II, Fig. 6B). The ORD parameters of n-hexanoyl, n-octanoyl and n-decanoyl asl-caseins

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446

L. IRONS et al.

(Table II) and the intensities of the Cotton effect troughs at 233 nm (Fig. 6B) all show a small increase in a-helix content compaied to tile unmodified protein. The most obvious effect of aeylation or succinylation on the ORD parameters of asl-casein B is to produce changes in the value of a0 (Table II). These changes are similar to those found for the corresponding derivatives of/~-easein A (refs I I , 12) and the order of the a 0 values of the modified proteins shown in Table I I follows closely the aggregate size order found from sedimentation studies. The more negative a 0 values of succinyl and acetyl asl-casein and of asl-casein B in guanidine hydrochloride compared with the native protein are probably due to dissociation of the asl-casein B aggregates, with the consequent exposure of previously buried groups to the aqueous solvent, plus the loss of some a-helix content. This is in agreement with other work, which has shown that succinylation of an unspecified genetic variant of asl-casein 1° and dissociation of asl-casein C at low protein concentrations a4 leads to more negative values of %. Conversely, the low a 0 values of the n-hexanoyl, n-octanoyl and n-decanoyl derivatives compared with that of unmodified as~-casein B m a y be caused by the structure of the aggregates, in which protein peptide bonds are located in a strongly hydrophobic environment. In general, transfer of peptide groups from a polar to a less polar environment reduces the value of a 0 (ref. 35)- The small increase in the (zhelix content of these derivatives m a y also contribute to the observed changes in a 0. At 25 °C, as~-casein B gives a good high lesolution NMR spectrum (Fig. 7A), although it is not identical to the theoretically computed spectrum for the equivalent random coil a6. This suggests a high deglee of flexibility compared to an ordered globular protein. The acetyl and succinyl derivatives give NMR spectra similar to aslcasein B but with additional well resolved resonances at 8.0 3 and 7.6o 3, corresponding to the unhindered CHACO- and -CH2CO- groups (Fig. 7 B, E). The spectra also show a slightly enhanced resolution of aromatic and methyl proton resonances. The NMR spectrum of propionyl asl-casein (Fig. 7 C) is similar to that of acetyl asl-casein but has a resonance from the propionyl methyl group at 8. 9 T as well as the -CH2COresonance at 7.6o 3. In all these spectra, there is evidence that substituent groups are in unhindered contact with the solvent, and in certain cases enhanced resolution is consistent with disruption of structure leading to dissociation and unfolding of polypeptide chains. The NMR spectrum of n-butyryl as~-casein is similar to those of the acetyl and propionyl derivatives, but it is not so well resolved (Fig. 7D). In contrast, there is much less resolution in the NMR spectra of ~-hexanoyl, n-octanoyl and n-decanoyl asl-caseins (Fig. 7 F, G, H). With n-decanoyl asl-casein, there is little evidence of the resonance at 8.75 3, which corresponds to the methylene groups of the substituent n-alkyl side chain. In 8 M !2H4]urea, n-decanoyl a~-casein shows a slight resolution of this resonance (Fig. 7I), but it only becomes fully evident in a spectrum obtained in trifluoro[~H?acetic acid (Fig. 7J)- This also is the case for the n-hexanoyl derivative. From these direct observations, it is clear that the highly aggregated, hydrophobic derivatives of as~-casein B must have a structure in which the substituent alkyl side chains are immobilised by inter- or intramolecular interactions which are not present in the unmodified protein. This confirms similar conclusions derived from the sedimentation studies. In conclusion, our previous studies with fl-casein A (refs I I , 12) and the present work with asrcasein t3 has shown that an appropriate choice of either partial or corn-

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p l e t e a e y l a t i o n or c a r b o x y a c y l a t i o n c a n b e u s e d t o c o n t r o l a s s o c i a t i o n b e h a v i o u r a n d the structural organisation of the aggregates. This implies that with these ploteins, a specific c h a r g e - h y d r o p h o b i c b a l a n c e c a n b e i m p o s e d t o p r o d u c e p r e d i c t a b l e s t r u c t u r a l a n d s e d i m e n t a t i o n effects. W h i l e a c y l a t i o n a n d c a r b o x y a c y l a t i o n a p p e a r t o b e g e n e r a l l y u s e f u l i n t h e s e cases, i t is n o t y e t k n o w n if t h e s e t e c h n i q u e s will p r o d u c e t h e same effects with globular proteins which do not associate in the native state.

REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23

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