THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 240, No. 3, March 1965

Printed in U.S.A.

Molecular Weight, Optical Rotation, and Tryptic

Susceptibility Studies on Modified Fetuins*

JACOB A. VERPooRTE,t WILLIAM A. GREEN,~ AND CYRIL iVl. KAY

From the Department of Biochemistry, University of Alberta Medical School, Edmonton, Alberta, Canada

(Received for publication, September 25, 1964)

The Lul-glycoprotein present in calf fetal serum, first described by Pedersen (1) and termed fetuin, may be isolated by ammo- nium sulfate fractionation (2) as well as by ethanol precipitation in the presence of Ba++ and Zn+f (3). The amino acid and carbohydrate composition has been determined for salt-pre- pared fetuin by Fisher, O’Brien, and Puck (4) and for the ethanol material by Spiro and Spiro (5). The percentage composition in each case is very similar, although differences were reported for the sialic acid content. This feature is re-examined in this study, and some additional molecular and structural properties of fetuin are also reported, following partial or essentially com- plete removal of the sugar acid.

A dramatic difference between s&-prepared and ethanol- prepared fetuins, from the same serum, was observed in terms of their behavior to proteolytic attack by trypsin and a-chymo- trypsin (6). The salt material was resistant to attack, confirm- ing the finding of Fisher, Puck, and Sato, (2), while the ethanol material was rapidly digested, as previously noted by Spiro (7). On the basis of this observation, it was concluded that the ethanol procedure for fetuin isolation results in a molecule with an altered secondary structure interpreted in terms of a loss cf helix content and trypsin resistance (6) and an increase in effective volume (8).

The stability of salt-prepared fetuin to proteolytic attack resembles that of the al-acid glycoprotein, orosomucoid, which, like fetuin, contains a high content of sialic acid, the removal of which results in proteolytic suscept.ibility (9). Optical rotatory dispersion data in the visible region suggested a low helical content for fetuin, in contrast to observations on the al-acid glycoprotein by Schmid and Kamiyama (10). Since p&al or essentially complete removal of sialic acid from fetuin results in an apparent loss of secondary structure, on the basis of visible optical rotatory dispersion measurements, it was felt desirable to confirm this observation by ultraviolet optical rotatory meas- urement.s as well. In addition, the effect of the removal of sialic acid on the molecular weight and tryptic susceptibility of fetuin has been investigated.

The association reaction of fetuin in the pH region near its isoelectric point (8) has been studied in more detail as well, along

* This investigation was supported by research grants from the Canadian Muscular Dystrophy Association, the Life Insurance Medical Research Funi, the-Canadian Medical Research Council and the National Institutes of Health (Grant AM-06287).

t Postdoctoral Fellow of the Canakan Muscular Dystrophy Association.

$ Predoctoral Fellow of the Canadian Muscular Dystrophy Association. Present address, Department of Medical Biochemis- try, University of Birmingham, England.

with the optical rotatory properties of the material at this low pH. Since heat treatment of salt-prepared fetuin resulted in ready tryptic digestion of this protein (6), some molecular and optical rotatory properties after heating, as well as aft’er partial removal of the sialic acid, are also described.

EXPERIMENTAL PROCEDURE

Protein Preparation-Two distinct commercial (NH&SO4 preparations were used in this st.udy: one obtained from the Colorado Serum Company, Denver, and the other from the Commonwealth Serum Laboratories, Melbourne, Australia. The detailed procedures for the preparation of these materials have been described previously (2, 11). Preliminary separation of the 18 S component was achieved by centrifugation (8, 11); the material was dialyzed free of salt and freeze-dried. Protein concentrations were determined by ultraviolet absorption at 278 rnp with Eit, values of 4.5 and 4.8 for the Australian and Colorado materials, respectively. These were determined on accurately weighed samples of the preparations, corrections being made for moisture content. The trypsin used in this study was purchased from Worthington Biochemical Corpora- tion, whereas the neuraminidase was obtained from Behring- werke A.G., Marburg, Germany.

Removal of Xialic Acid from Fetuin-Removal of sialic acid may be performed by acid treatment (3) as well as by neuramini- dase digestion (4, 6). In our study, the acid hydrolysis was performed at pH 2 and 4” for several days, and samples were abstracted for study over several time intervals during this period. The neuraminidase treatment was performed at 25” in a 0.01 M acetate buffer, pH 5.5, which also contained 9 g of NaCl per liter. To 25 ml of a 1% fetuin solution were added 2 ml of neuraminidase solution containing a total of 200 units. Each neuraminidase unit corresponded to 1 pg of N-acetyl- neuraminic acid released during 15 minutes at 37” and pH 5.5 from a glycopeptide substrate in the above solvent, which also contained 1 g of CaClz per liter. The mixture was placed in a dialysis bag and immersed in 220 ml of buffer. The course of the reaction could be followed directly on 0.2-ml samples with- drawn from the dialysate, or on 0.2 ml of a lo-fold dilution of the reaction mixture, taken from the dialysis bag. The amount of liberated sialic acid was determined by the thiobarbituric acid assay method of Warren (12). For acid-hydrolyzed material, the sialic acid contents of the different fetuin preparations were also obtained by the method of Warren, after hydrolysis of the material in 0.1 N H&04 in a boiling water bath.

Proteolysis of Fetuin-The rate of proteolytic attack of fetuin by trypsin was followed in the pH-stat at pH 8.0 and 25”. TO

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3 ml of a 0.33% fetuin solution in 0.1 M NaCl, was added 0.1 ml of 0.17, trypsin, freshly dissolved in low3 M HCl. The pH was adjusted to 8 by the addition of 0.1 M KOH. The rate of proton release was recorded as equivalents of standard base (approxi- mately 2 X 10-s M NaOH) added during the reaction.

Sedimentation Velocity Measurement-The Spinco model E analytical ultracentrifuge was used at speeds of 59,780 r.p.m. and at temperatures near 20”. The usual corrections to sBo,W were then made (13).

Molecular Weight DeterminationsMolecular weights were determined by the method of Archibald (14), as described by Schachman (13). Various speeds were used during the approach to sedimentation equilibrium runs, depending on the degree of association. The protein concentrations were generally -in the range of 0.75 %. A bar angle of 75” was employed in the schlieren studies, and molecular weights, for comparative purposes, were computed from Archibald data at the meniscus position only.

Optical Rotatory Dispersion- Optical rotation measurements over the wave length range of 340 to 600 rnp were obtained by means of a Rudolph model 260 automatic recording spectro- polarimeter. The light source used was a tungsten lamp, and the solutions were placed in a cell of 5-cm path length. A sym- metrical angle of 5” was used throughout the measurements. The data were treated by the method of Yang and Doty (15) which involves plotting -Xz[a] versus -[a!] to determine X,, and also by the Moffitt treatment wherein [m’]k[(Xz - X0z)/X02] is plotted against k?/(X2 - Xo2). The dispersion parameters, a0 and b0 of the Moffitt equation, were obtained from the ordinate intercept and slope of the plots, respectively (16). Mean residue rotations were computed on the basis of an average residue weight of 110, whereas the dispersion of the refractive index, n,

of the solvent used was assumed to be the same as that of pure water. A value of 205 rnp was assigned to X0 for reasons given elsewhere (6).

In a recent paper, Shechter and Blout (17) propose a new analysis of optical rotatory dispersion data in the region of visible and near ultraviolet wave lengths ( >260 mp). Their method is not only useful for the determination of the helical content, of proteins in solution, but also provides a basis for the differentiation of the a-helix from other protein conformational forms such as p structures. The data are normally plotted in the form of a modified two-term Drude equation, and a good fit is obtained by fixing one term at X1 = 193 rnp and choosing

the other at XZ = 225 mp. By this method, [m’]h[(X2 - X&)/ X&] was plotted against &?/(X2 - k&), yielding a straight line relationship from which the new dispersion parameters A (epp25 and A (ap)193 were evaluated. The slope of such a plot is equivalent to A (ap)?25 x (X2252 - X1,,2)/X1932 whereas the ordinate intercept is A (ap)~~~ + A (mp)22~ x X22~~/Xd. The A caO) values are directly related to the Cotton effects, and therefore to the a-helical content.

Optical rotation data in the wave length region of 215 to 320 mp were obtained from a Rudolph model MSP4 manual spectro- polarimeter with an Osram xenon lamp as the light source. A l-cm cell was used in the wave length region of 250 to 320 rnp and a O.l-cm cell from 250 to 215 ml. A symmetrical angle of 2” was used throughout the measurements, and the concentra- tion of the protein solutions examined was 0.03 to 0.04%. The helix content of the proteins was estimated by the method of Simmons et al. (18) with the use of [m’]233 values for poly-L- glutamic acid of -15,000” for the 100% helical form and -2,000” for the random coil (19). The helicity of the proteins was calculated by simple linear interpolation.

RESULTS

Visible Optical Rotatory Dispersion Measuremen!s on Colorado and Australian Fetuin-Table I lists the dispersion characteristics (X,, a,,, b,J for the two commercial fetuin preparations employed in this study at several pH values and also under some heating conditions. The Moffitt plots were linearized by employing a X0 value of 205 mp, as previously discussed. For any one preparation, the rotational parameters are essentially the same, irrespective of the pH. On the other hand, when one compares the two preparations at any one pH value, there are small but significant differences in the measured quantities. Also in- cluded in Table I are the rotatory dispersion parameters A (crP)225 and A cap)rg3, evaluated from the Shechter and Blout analysis. The significance of these will be elaborated in the discussion. Examples of representative plots of the Shechter-Blout type for both preparations are shown in Fig. 1. Fig. 2 represents a plot of A (ap)225 versus A cap)rg3, deduced from the above analysis for all our experimental data. The experimental points recorded in this study do not fall exactly on the linear curve of Shechter and Blout (17); however, they do fall more closely on this curve than those of pepsinogen and fl-lactoglobulin, cited by Shechter and Blout as nonhelical (17).

TABLE I

Optical rotatory dispersion data on fetuin in 0.1 M NaCl*

Material

Australian fetuin

Colorado fetuin

-

-

PH

7-8 f20° 229

3.5 f20” 226 2.5 f20° 228

- 108 -126 -122

7-8 f20° 230 - 140

3.5 f20” 234 -141

2.5 f20” 232 -136 5.0 Got 224 -80 7.5 6Ot 230 - 104 7.5 sot 212 -54

Temperature ?.,fZrn/.l Eo I_ I

-L

a0 A( A(.,)226

-419 +26 -368 -407 +156 -480 -427 +56 -401

-320 +208 -425 -331 +256 -440

-365 +76 -368

-424 -166 -240 -365 -t86 -368 -401 -169 -212

* These measurements were completed within an hour after equilibration of the solution at the desired pH. Over this time inter- val, there was a negligible sialic acid release in those solutions at acid pH values.

t Samples were heated at 60 and 80” for 20 minutes in a water bath and measured at room temperature.

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1158 Properties of ModQied Fetuin Vol. 240, No. 3

-500 0 +A0 Atop) 193

I 2 3 4 5 6 7 6

A2222

-ix lo-’ FIG. 2. Plot of Ac,,jzzs versus A(tip)~9~ for the visible optical rota-

tory data presented in Table I, together with the experimental curve of Shechter and Blout (-), represented by the equation

FIG. 1. Shechter-Blout analysis for Australian (X-X) and A(,,,225 = -0.55A (ap)193 - 430, and their plotted points for p-lacto- Colorado (e-0) fetuins. Concentration of protein solutions, globulin (A) and pepsinogen (B). X, Colorado fetuins; 0, Aus- 0.59Zo in 0.1 M KCl, pH 7. tralian fetuin.

Material

Australian fetuin

Colorado fetuin

TABLE II Properties of fetuin after partial removal of sialic acid by neuraminidase

-

.-

lhe of neuramini- dase reaction

hrs

2 4 6

8 12 24

40 241

2 4 6

26 40

241

7-

1

_-

-

Moles sialic acid/ mole fetuin’

8.9 43,000 -3,730 8.5 49,000 -3,515 8.1 54,000 -3,080 7.8 5G,OOO -2,620

7.4 56,000 -2,090 6.4 56,000 -2,050 3.7 56,000 -2,040

1.8 56,000 -2,040 8.8 44,000 -3,410

%

13.3 11.6

8.3 4.8 0.7

Negligible Negligible Negligible

10.9

0.2

0.2 1.2 1.75 1.85 1.80

1.80 1.90 0.6

10.4 48,300 -3,780 13.7 0.2

9.9 56,000 -3,470 11.3 0.9

9.5 64,000 -2,910 7.0 1.95 9.0 67,000 -2,080 0.6 2.45

3.7 67,000 -2,010 Negligible 2.50 1.1 69,000 -1,940 Negligible 2.60

10.2 49,000 -3,450 11.2 0.9

Molecular weight Im’l2aa

.-

Helix content based on [m’lzas

- 1 3onds cleaved/l0 min by trypsin

* Based on an average molecular weight of 45,000. f Solutions of fetuin in buffer, pH 5.5, were stored at 25” for 24 hours.

Properties of Desialicized Fetuin-To avoid the possibility of additional alterations occurring in the fetuin molecule in acid media, we generally performed the removal of sialic acid by neuraminidase treatment. In those cases in which appreciable changes occurred as a result of enzymic treatment, the results were confirmed by the use of acid. When fetuin was treated with neuraminidase, no further release of sialic acid could be observed after approximately 40 hours of treatment. The total amount of sialic acid liberated differed for the two fetuin prepa- rations at comparable time intervals as noted in Table II. Removal of 1.4 moles of the total sialic acid, for Colorado mate- rial, and about 1.5 moles for the Australian material, resulted in a complete loss of helical content, as deduced from ultraviolet optical rotatory measurements based on the Cotton trough at 233 rnp (see Fig. 3). Table II refers to neuraminidase treat- ment, but the figures obtained by acid treatment are identical.

Noteworthy is the increase in weight average molecular weight

that parallels the initial release of sialic acid (Table II). How- ever, the removal of sialic acid, beyond approximately 1.2 moles per mole of fetuin, does not appear to alter further the weight average molecular weight. The weight average molecular weights recorded (56,000 for Australian fetuin and about 67,000 for Colorado material) could possibly be rationalized on the basis of an equilibrium mixture of monomer and dimer (see Fig. 4a). The association forces are readily broken by the addition of 5 M guanidine.HCI.1

Table II also records the ready digestibility of fetuin by trypsin after the removal of sialic acid. The release of one sialic acid per mole of protein results in rapid digestion for both Australian and Colorado materials.

1 A molecular weight of -45,000 was recorded for desialicized fetuin in 5 M guanidine.HCl and phosphate buffer, pH 7.8. The partial specific volume, P, was assumed to be identical with that of the native material, viz. 0.70 ml per g.

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March 1965 J. A. Tierpool-te, W. A. Green, and C. M. Kay 1159

Association of Fetuin at Low pH ValuesFetuin solutions were made up in 0.1 M NaCl, and the pH values adjusted with the aid of a Radiometer TTTl pH meter, with glass electrodes calibrated against standard buffers. The optical rotation experiments were completed within 1 hour after the preparation of the

3

2

I

0

x ? E -I

Y

-2

-3

2( . .

I m 220 240 260

Xinmp

FIG. 3. Rotatory dispersion between 220 and 2GO rnp for native (X-X) and desialicized (8-O) Colorado fetuin.

FIG. 4. Representative sedimentation-velocity patterns at 59,780 r.p.m. at bar angle 50” and 40 minutes after reaching top speed for: a, desialicized fetuin in phosphate buffer, pH 7.8, I = 0.2; b, native fetuin in 0.1 M KCl, pH 2.5; c, desialicized fetuin in 0.1 M KCl, pH 2.5; cl, native fetuin, heated to BO”, in 0.1 M KCl, pH 5.0; e, native fetuin, heated to 60”, in 0.1 M KCl, pH 7.5;f, desiali- cized fetuin, heated to 70”, in 0.1 M KCl, pH 5.0.

TABLE III Molecular weight and helix content of fetuin at low pH

Material PH bdlraa Helix

%

Australian fetuin 7-8 -3,680 12.9 3.5 -3,730 13.3 2.5 -3,750 13.4

Colorado fetuin 7-8 -3,830 14.0 3.5 -3,950 15.0 3.0 -3,680 12.9 2.5 -3,850 14.2 2.0 -3,670 12.8

Molecular weight

43,300 102,000 100,000

48 ) 400 116,000

132.000

solutions, and the visible and ultraviolet results are given in Tables I and III, respectively. The helical contents of either Australian or Colorado fetuin are not appreciably altered over the pH range 8 to 2, on the basis of either the Cotton amplitude at 233 rnk or the b. and X, values.

The weight average molecular weights of fetuin, in acid media, are also recorded in Table III, from which it may be seen that association has occurred to the level of -100,000 for Australian fetuin and -130,000 for Colorado material. Removal of sialic acid did affect the association slightly, a value of -90,000 having been recorded for essentially completely desialicized Colorado material at pH 2.5 (see Figs. 4b and 4~). An equilib- rium system of dimer-trimer might be an explanation for this association at low pH. In accordance with previous studies (8), it was found that the reaction was reversible by readjusting the pH. No association could be detected in 5 M guanidine.HCI; in this solvent system, a molecular weight of 35,000 to 40,000 was obserl ed for fetuin at pH 2.5.

E$ect of Heat Treatment on Molecular Weight, Optical Rotatory

Properties, and Tryptic Resistance of Fetuin-Solutions of fetuin, and material from which sialic acid had been removed, in 0.1 M

NaCl at different pH values were heated in a thermostat for 20 minutes, after which they were cooled in tap water. In accordance with the observations of Spiro (3), no visible coagu- lation could be detected after heating, but ultracentrifuge experi- ments indicated aggregation with two distinct peaks in the schlieren patterns, with SZO+ values of -3 and 6 to 7 S (see Figs. 4d and 4e). As noted in Table IV, the association is in- creased by temperature, while the pH also affects it. It appears that the removal of sialic acid before heating enhances the aggregation (Fig. 4j). There are indications, on the basis of molecular weight studies on lyophiliaed heated material, and heated material dissolved in 5 M guanidine .HCl, that the heat aggregation process is reversible.

Table V records that the helix content of fetuin, on the basis of [m’]233 values, decreases rapidly on heating, and does not seem to be affected by the pH of the solution at pH values of 4 and 8. Essentially the same [m’]233 values were recorded for fetuin at pH values of 5, 6, and 7 under comparable heating conditions. Since the decrease in helical content of fetuin is irreversible, optical rotation measurements could be made at room temperature. The helix change seems to precede the observed aggregation, because essentially 0% helix was found at much lower temperatures than were required for maximum aggre- gation. A similar trend towards decreasing helicity was ob-

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1160 Properties of ModiJied Fetuin Vol. 240, No. 3

TABLE IV

Effect of heat on molecular weight of Colorado fetuin

Moles of sialic acid/mole of fetuin* PH

10.4 5.0 60" 155,000 10.4 5.0 70 215,000 10.4 5.0 80 405,000

10.4 6.5 70 100,000 10.4 7.5 70 80,000 10.4 7.5 80 124,000

10.4 8.0 80 70,000

8.7 5.0 70 260,000 5.1 5.0 70 290,000

2.9 5.0 70 370,000 1.1 5.0 70 410,000

Temperature Molecular weightt

* Based on a molecular weight of 45,000. Sialic acid was re- moved by neuraminidase treatment, but acid treatment gave identical results.

t The weight average molecular weights were computed from comparative runs at 48 and 64 minutes at maximum speed of 8225 r.p.m.

TABLE V

E$ect of heat and pH on helix content and trypsin resistance of Colorado fetuin

Tempera- ture

30" 40 50 60

70 80

-3670

-3740 - 2990 -2150

pH 4.0 -

%

12.9 13.4

7.6 1.1

Negligible

Negligible

pH 8.0

T -3790 -3640

- 2990 -2150

Helix

%

13.8 12.6

7.6 1.1

Negligible Negligible

onds cleaved/l0 nin by trypsin*

1.35 2.5

2.6 2.6

* The rate of tryptic digestion is given for fetuin heated at pH 8.0, but was found to be identical for fetuin heated at pH 5.0.

served in heated fetuin solutions, on the basis of visible optical rotatory measurements (see Table I).

Some experiments performed on the tryptic resistance of heated fetuin solutions indicated that the susceptibility to this enzyme parallels the decrease in helical content, and that the aggregation does not affect. the trypsin digestion (Table V).

DISCUSSION

It has been shown previously by Green and Kay (6) that the highly charged sialic acid groups contribute appreciably to the maintenance of the secondary structure of fetuin. This study clearly confirms this sialic acid function. The release of as little as 1 mole of the sugar acid per mole of fetuin results in a completely altered molecule; the secondary structure changes, the weight average molecular weight increases, and the trypsin resistance of fetuin is lost..

Similar changes were observed when a fetuin solution was heated. In this case, the weight average molecular weight became much higher, the actual value being a function of the pH of the solution. At neutral or weakly basic pH values, heat aggregation was far less than at weakly acid pH values, where

two distinct peaks were observed in the ultracentrifuge, with sZo,w values of -3 and 6 to 7 S, respectively. When fetuin solutions were heated to 80” at pH 5, only a trace of sialic acid was liberated, and none whatsoever at higher pH values. This might indicate that the liberation of sialic acid increases the aggregation; indeed, large amounts of aggregates were formed in heated solutions of partially desialicized material. At pH values between 4 and 8, the alteration in secondary structure following heating is a function of temperature only, and therefore is not related to the release of sialic acid. It is probable that no sialic acid groups participate in the association which occurs following partial removal of some of these groups, or after heating.

The reversible association at low pH values also appears to occur without sialic acid group involvement. The latter associa- tion, however, takes place without significant changes in second- ary structure. It remains to be seen whether at pH 2, the lowest pH used in our optical rotatory dispersion studies, the negative charge of sialic acid is completely neutralized, since titration studies suggest a pK of 2.3 t,o 2.4 for these groups (3). The effect of heat and acid, respectively, on native fetuin emphasizes the need to avoid exposure of the protein to extremes of these conditions and underlines the importance of the history of the blood sample from which the fetuin is isolated. The difference in history of the blood sample might also be reflected in slight differences in the chemical composition (sialic acid content) and physical properties of various preparations (e.g. salt-fractionated material versus ethanol material and Colorado versus Australian fetuin).

Heat and acid treatments of salt-fractionated fetuin both result in an alteration in secondary structure, which might be taken to represent a decrease in helix content. Although it is yet unknown if, and in what way, the presence of about 20% oligosac- charide affects the optical rotatory dispersion measurements of fetuin, some influence of the sugar units might be reflected in the X0 value chosen to obtain linear Moffitt plots. In accordance with the finding of Green and Kay (6), linear plots were obtained with a X0 of 205 rnp, a value which differs widely from that employed for al-acid glyco-protein, viz. 220 rnp (10).

Dispersion measurements in the ultraviolet region clearly indicated the features of helical structures. To deduce the helix content from [m’]233 values, we assigned the values of -2,000” for the random conformation and -15,000” for 100%

helix (19). In our studies, the -2,000” value was confirmed for fetuin in the random conformation, achieved by heating or desialicization, and a significantly lower value has never been detected.

An analysis of the dispersion data from experiments in the visible wave length region by the method of Shechter and Blout (17) gave helix parameters, 9 (ap)193 and A cap12z5, which do not exactly fit the linear relation shown by the aforementioned authors. The results, however, were conclusive enough to assume a helix structure in fetuin. The relation between the or-helix parameters calculated for aqueous systems is given by A (q)225 = -0.55 A&?)193 - 430 (17). These parameters were shown to be influenced appreciably by the dielectric constant of the solvent and a new intercept of -280 was found for low dielectric constant solvents (20). Coincidentally perhaps, our data for these parameters, as given in Table I, fit much better a linear relation with an intercept of -330, the reason for which is still obscure. It was therefore concluded that the helix content of fetuin could be calculated from the equation; H = [A (ap)193 -

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March 1965 J. A. Verpoorte, W. A. Green, and C. M. Kay 1161

A(ap)225 + 650]/55.8, a relationship which was independent of solvent, to a first approximation (20).

However, helix contents calculated with this formula are con- siderably higher than those obtained from [&I233 values. It might be erroneous, however, to calculate “helicity” from [v&~ values because of the possible influence on the rotation by the close to 20% carbohydrate. The values given should there- fore not be taken too literally. The helical content can also be evaluated from the parameter 60, obtained from a Moffitt plot. Here, as in the analysis by the method of Shechter and Blout, the tentative assumption must be made that the presence of carbo- hydrate, while possibly influencing the over-all rotation of the molecule, does not interfere with the dispersion of the peptide chain. The helix contents calculated from bo, assuming limits of -700” and 0” for 100% helix and random coil, respectively, are also somewhat higher than those obtained from [v&~, values of 18 to 20% helix having been calculated. We have therefore taken a helix content of 15% in salt-precipitated fetuin as the mean value.

The sialic acid groups are of importance in at least two func- tions in fetuin, viz., the secondary structure of the molecule and its resistance to tryptic attack. Along these lines, they find particular appeal, following the conclusions of Spiro (3, 7) that there are three similar carbohydrate moieties in fetuin, distrib- uted along the peptide chain, each with branched structures con- taining peripherally located sialic acid groups. In this fashion, it is evident that a sheath of negative charge could be provided by a suitable disposition of the sialic acid groups around the protein core of the molecule. The repulsive field so generated would prevent aggregation and would impede the approach of any molecule or charged group that lacked a net positive charge. In view of the readiness with which fetuin aggregates when this negative charge is only partly removed, one is tempted to con- clude that whatever its biological function is, it may be related to the integrity of its charge envelope. This is, of course, the case for its resistance to attack by trypsin.

SUMMARY

The associative properties of fetuin, as well as its tryptic susceptibility and loss of secondary structure, following the release of as little as one sialic acid group, have been established. Heating of a fetuin solution results in partial aggregation, two distinct peaks being observed in the ultracentrifuge, at weakly acid pH. Heating also results in a loss of secondary structure and increase of trvnsin suscentibilitv.

The tryptic digestibility of fetuin parallels the loss of secondary structure and is not related to aggregation or release of sialic acid.

The association of fetuin at low pH has been reinvestigated and appears to occur independent of the sialic acid and helix content.

A careful analysis of optical rotatory dispersion measurements in the visible and ultraviolet regions of the spectrum indicates the presence of helix structure in fetuin. Calculations by several methods demonstrate approximately 15% helix in the molecule.

Acknowledgments-We are indebted to J. Durgo, A. Keri, and K. Oikawa for their skilled technical assistance. We are also grateful to Mr. R. J. Swindlehurst of the Chemistry Depart- ment, University of Alberta, for kindly running the optical rotatory dispersion curves in the Rudolph model 260 recording spectropolarimeter,

1. 2.

3. 4.

5. 6. 7. 8.

9. 10. 11.

12. 13.

14. 15. 16.

17.

18.

19.

20.

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