THF: JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 246, No. 13, Issue of July 10, pp. 41964205, 1971 Printed in U.S.A.

The Subunit Structure of Horse Spleen Apoferritin

I. THE MOLECULAR WEIGHT OF THE SUBUNIT

(Received for publication, December 1, 1970)

C. F. A. BRYCE* AND R. R. CRICHTON$. 5

From the Department of Biochemistry, University of Glasgow, Glasgow, W.2. Scotland

SUMMARY

The subunit molecular weight of horse spleen apoferritin has been determined by polyacrylamide gel electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate, gel filtration on columns of agarose in 6 M guanidine hydro- chloride, and by sedimentation equilibrium under dissociat- ing conditions in the analytical ultracentrifuge. The results obtained indicate that the polypeptide chain of horse spleen apoferritin has a molecular weight of 18,500, which is in marked contrast to the previously determined value. The tryptophan content of the protein was estimated spectro- photometrically and, after modification, as 2-(2nitrophenyl- sulfenyl)tryptophan. The value of 2.08 residues per 18,500 daltons is also in disagreement with literature values.

These observations suggest that the undissociated horse spleen apoferritin molecule may consist of 23 to 25 subunits. For reasons of symmetry a value of 24 would seem most probable.

The molecular weight of human and horse transferrin was also determined and found to be 76,600.

Ferritin, the principal iron storage protein of mammals, was first isolated and crystallized by Laufberger in 1937 (1) and since this time many attempts have been made to characterize the intrinsic structure of the protein.

Both the electrophoretic and immunological studies of Mazur and Shorr (2) and the electron microscopic studies of Farrant (3) clearly indicated that the iron, in the form of micelles of ferric hydroxyphosphate, lies within the protein coat and not, as was originally thought, on the surface (4). The iron of ferritin can be reduced (5-7) and removed by dialysis to give, without de- naturation of the protein, a protein shell (apoferritin) which is roughly spherical as determined by electron microscopy of nega- tively stained preparations @-lo), low resolution x-ray diffrac-

* Recipient of University of Glasgow Faculty of Science Stu- dentship during part of this project and recipient of a Research Studentship from the Science Research Council.

$ To whom inquiries and reprint requests should be addressed. $ Present address, Max-Planck-Institut fur Molekulare Ge-

netik, Abt. Wittmann, Berlin 33 (Dahlem), Ihnestrasse 63, West Germany.

tion measurements on wet crystals of the apoprotein (ll), and small angle x-ray scattering (12-14).

Harrison deduced from higher resolution x-ray diffraction studies that apoferritin was a roughly spherical molecule possess- ing 432-point group symmetry and consisting of 24n identical subunits and suggested that the most likely arrangement would be a snub-cube with 24 vertices (15). However, further physical and chemical studies (16-20) led to the conclusion that the num- ber of subunits per molecule of undissociated apoprotein was 20 which is also a value suggested by Harrison from more detailed examination of the diffraction patterns (11) and that these quasi- equivalent subunits would be situated at the vertices of a pentagonal dodecahedron.

In an attempt to clarify this anomaly we decided to reinvesti- gate the values determined for the molecular weights of both undissociated and dissociated horse spleen apoferritin. The previous value for the undissociated moiety ranged from 430,000 to 465,000 (15, 21, 22) while that for the subunit was determined to be 25,000 to 27,000 (19).

Two empirical techniques for the determination of the molecu- lar weights of proteins and polypeptide chains have recently been devised which, with few exceptions, can afford a precision of 5 to 6% (23, 24). Essentially these techniques are based on molecu- lar sieving which is performed in the presence of denaturing media so that the molecular volume of the fully reduced and un- folded polypeptide chain is dependent almost solely on the num- ber of residues and not on their spatial organization. Fish, Mann, and Tanford (23) determined the partition coefficient of a wide range of proteins and peptides on columns of agarose beads equilibrated with 6 M guanidine hydrochloride, and found a smooth sigmoidal curve over the range 1,000 to 80,000 daltons. The other approach, the reliability of which has been exhibited for well characterized polypeptide chains over a very wide range of molecular weight (25, 26), is to determine the electrophoretic mobility on polyacrylamide gels in the presence of the anionic detergent sodium dodecyl sulfate (24, 27-29).

It was also decided to determine the molecular weight of the undissociated and the dissociated apoferritin by sedimentation equilibrium in the analytical ultracentrifuge.

The value for the apparent molecular weight of the subunit obtained from these procedures is in disagreement with the previously determined values and this is discussed in relation to the number of subunits present in the undissociated apoferritin molecule.

A preliminary report of this work has appeared (30).

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of July 10, 1971 C. F. A. Bryce and R. R. Ctichton

EXPERIMENTAL PROCEDURE

Materials

The proteins used in this study were obtained from Mann: apoferritin (horse spleen) and catalase (bovine liver) ; from Worthington: alcohol dehydrogenase (liver), cr-chymotrypsin (bovine pancreas), chymotrypsinogen A, leucine aminopeptidase, ovalbumin, and trypsin; from Sigma: carboxypeptidase A (bovine pancreas), creatine phosphokinase (rabbit muscle), cytochrome c (horse heart), hemoglobin, insulin, lysozyme (egg white), pepsin, and ribonuclease A (bovine pancreas) ; from Koch-Light Labora- tories Ltd., Colnbrook, England: ferritin (horse spleen), papain (papaya latex), and transferrin (horse, pooled plasma) ; from Armour Pharmaceutical Company Ltd., Eastbourne, England: albumin (bovine plasma) ; from Pentex: apoferritin (horse spleen), -y-globulin (porcine); from British Drug Houses Ltd., Poole, England: myoglobin (horse heart) ; and from Ciba Laboratories, Ltd., Horsham, England: synacthen (0 l-24 corticotropin).

Analytical grade NaH2P04. 2Hz0, Na2HP04, ammonium persulfate, acrylamide, N, N’-methylene bisacrylamide, N, N, N’ , N’-tetramethyl-1 ,2-diaminoethane, sodium dodecyl sulfate (specially pure), bromphenol blue, guanidine hydrochloride, tryptophan, activated charcoal, glycine, urea, 2-nitrophenylsul- fenyl chloride, toluene, methanol, formic acid (98 to loo%, sp. gr. 1.22), phosphoric acid (88%, sp. gr. 1.75), and acetone were obtained from British Drug Houses, Ltd., Poole, England. 2-Mercaptoethanol, cyanogen bromide, and ninhydrin were pur- chased from Koch-Light Laboratories Ltd. Dimethyldichloro- silane was obtained from Bio-Rad Laboratories, Richmond, California. Naphthalene black 10B was obtained from George T. Gurr Ltd., London, England. Blue dextran 2000 and Sepharose 6B were obtained from Pharmacia.

Methods

SDS-Polyacrylamide Gel Electrophoresis

The proteins and polypeptides used in this study as molecular weight markers together with their molecular weights are listed in Table I. Of each protein, 0.5 mg was dissolved in 1 ml of 0.01 M sodium phosphate buffer, pH 7.0, 1% in SDS, and 1% in 2-mercaptoethanol and incubated at 37” for 4 to 5 hours. The solutions were then dialyzed for 16 hours against two changes of the same buffer containing 0.1% SDS and 0.1% 2-mercapto- ethanol. They were then diluted 1: 1 with 0.01 M sodium phos- phate buffer, pH 7.0, 0.1% SDS, 0.2% 2-mercaptoethanol, 0.005% bromphenol blue, and 20% glycerol. The 10% poly- acrylamide gels used in the electrophoresis contained 0.1% SDS and 0.1% 2-mercaptoethanol and these were cast in 15-cm gel tubes (long gels were used in an attempt to improve the separa- tion in the molecular weight range used). The diluted protein solution, 100 ~1 (corresponding to 25 pg of protein), was applied to each gel and 0.1 M sodium phosphate buffer, pH 7.0, 1% SDS was then carefully layered on top. This same buffer was used as the electrode buffer and electrophoresis was carried out at 20 ma per gel. After electrophoresis the gels were removed from the tubes by rimming with water and were then scanned with a Vitatron densitometer (Vitatron Instruments Ltd., Dieren, Netherlands), in order to determine the distance traveled by the blue marker dye (bromphenol blue) and also the length of the gel.

1 The abbreviation used is: SDS, sodium dodecyl sulfate.

TABLE I

Protein polypeptide chains used as molecular weight markers

Polypeptide Molecular weight Reference

Transferrin ............................... 76,600 Serum albumin. .......................... 68,000 Catalase. ................................ 60,000 r-Globulin, H chain. ..................... 55,000 Leucine aminopeptidase. ................. 53,000 Ovalbumin. .............................. 43,000 Alcohol dehydrogenase (liver). .......... 41,000 Creatine phosphokinase .................. 40,000 Pepsin ................................... 35,000 Carboxypeptidase A. .................... 34,600 Chymotrypsinogen A. ................. 25,700 -y-Globulin, L chain ....................... 23,500 Papain .................................. 23,300 Trypsin .............................. 23,000 Myoglobin ......................... .... 17,200 Hemoglobin. ........................... 15,500 Lysoeyme. ............................... 14,300 Ribonuclease ............................. 13,700 Chymotrypsin, H chain. ............... 13,000 Cytochromec .......................... 11,700 Chymotrypsin, L chain. ................ 11,000 Cytochrome c, CNBr peptide I., .......... 7,650 Insulin .................................. 6,000 Synacthen ................................ 3,360 Cytochrome c, CNBr peptide II. ......... 2,530 Cytochrome c, CNBr peptide III. ........ 1,540

31, 32 33 34 29 25 35 36 37 38 39 40 29 40 40 40 40 40 40 40 40 40 40 40 40 40 40

The gels were then stained for 20 min with staining solution (2.5 g of Amido black, 908 ml of 50% methanol, 92 ml of glacial acetic acid), and then allowed to destain for 30 hours in destaining solu- tion (7.5% glacial acetic acid and 5% methanol in water). They were then scanned in the densitometer to determine the distance traveled by the proteins and the length of the gel after staining. The electrophoretic mobility, defined as distance of protein migration X gel length before staining/distance of dye migration X gel length after staining, was calculated for each protein and a plot of logarithm of molecular weight versus electrophoretic mobility was constructed.

Agarose Gel Filtration

The molecular-sieving medium used in these experiments was Sepharose 6B with a nominal agarose content of 6%. The col- umn used was a Pharmacia chromatographic column (1.5 x 90 cm) which had been silanized before use. (Treatment with 5% (v/v) dimethyldichlorosilane-toluene at 60” for 2 hours.) This was packed to a height of 85 cm with the agarose gel, equilibrated with 6 M guanidine hydrochloride, pH 5.0. Extreme care must be exercised at the stage of equilibration to prevent degradation of the agarose beads. The gel was treated with a solution of 6 M

guanidine hydrochloride (solution of analytical grade guanidine hydrochloride heated to 60” with activated charcoal for 4 hours and filtered through Whatman No. 42 filter paper) and allowed to deaerate without stirring at room temperature for about 20 hours. The proteins and polypeptides were prepared for gel filtration by incubation in 6 M guanidine hydrochloride, pH 8.6, 0.1 M 2-mercaptoethanol for 6 hours at 37”. The proteins thus unfolded and fully reduced were then carboxymethylated by

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4200 Subunit Molecular Weight of Apoferritin Vol. 246, x0. 13

treatment with iodoacetic acid. The concentration of protein used in these studies was normally 30 to 40 mg per ml of the guanidine solution. Blue dextran (5 mg), 1 ml of tryptophan, and 3 to 4 drops of glycerol were then dissolved in 1 ml of the protein solution, and 0.2 ml of this sample was then applied to the column. The column effluent was passed through the flow cell of an LKB Uvicord II (LKB Instruments, London, England) monitoring at 280 nm and the effluent from this was collected in l-ml fractions in a BTL Chromofrac fraction collector (Baird and Tatlock Ltd., London, England), thus permitting us to read the absorbance at 630 nm (blue dextran) manually on a Beckman DB spectrophotometer (Beckman Instruments, Palo Alto, California). The flow of eluting solvent was maintained at a rate of 2 ml per hour by use of a solvent reservoir.

Analytical Ultracentrijugation

Undissociated Apojerritin-Lyophilized apoferritin was dis- solved in 0.01 M sodium phosphate buffer, pH 7.0, and the re- sultant solution was dialyzed for 16 hours against the same buffer at room temperature. The molecular weight determinations were performed over a protein concentration range of 0.075 to 1.0 mg per ml and a rotor speed of 3,600 rpm was used.

Dissociated Apojerritin-Since it is known that apoferritin can be dissociated into subunits by several protein denaturants it was decided to determine the molecular weight of the subunit ob- tained from different dissociating conditions.

Dissociation by 6 M Guanidine Hydrochloride-Lyophilized apoferritin was dissolved in 6 M guanidine hydrochloride, pH 7.0, and incubated at 37” for 4 hours. The solution was then dialyzed for 16 hours at room temperature against the guanidine hydro- chloride solution. The determinations were performed over a protein concentration range of 0.2 to 0.6 mg per ml.

Dissociation by Acetic Acid-Two volumes of glacial acetic acid were added to 1 volume of apoferritin solution (0.7 mg per ml) and the resultant mixture maintained at 0” for 1 hour. The solution was then dialyzed overnight against two changes of 0.01 M glycine-KC1 buffer, pH 3.0.

For the apoferritin subunit, the molecular weight determina- tions were performed with a rotor speed of 20,000 rpm.

Sedimentation equilibrium experiments were performed in a Spinco model E analytical ultracentrifuge (Beckman Instru- ments, Palo Alto, California) equipped with electronic speed control and regulated temperature control units. The use of the split beam photoelectric scanner accessory, in conjunction with a monochromator and the ultraviolet absorption optical system (41), permitted the recording of sedimentation patterns at 280 nm and made possible the use of very low concentrations of protein. Whole cell apparent weight average molecular weights (% app) were determined at 20”.

Quantitative Tryptophan Determination

Method I-Horse spleen apoferritin was incubated in a mixture of 6 M guanidine hydrochloride and 0.1 M 2-mercaptoethanol, pH 8.6, at 37” for 6 hours. The reduced apoferritin so obtained was carboxymethylated according to the procedure of Canfield and Anfinsen (42).

The fully reduced and carboxymethylated protein (20 mg) was dissolved in 2 ml of 50% formic acid. To 1 ml of the solution was added 0.5 ml of glacial acetic acid containing 2.5 mg of 2-nitrophenylsulfenyl chloride (43). The mixture was incubated at room temperature for 10 min and the modification reaction

was terminated by the addition of 100 ml of ice-cold acetone-l N hydrochloric acid (39: 1). The resulting precipitate was centri- fuged, washed with ice-cold acetone, and dried under reduced pressure over concentrated sulfuric acid. The modification of the cysteinyl residues was then reversed by incubating the puri- fied labeled protein with 0.1 N NaOH at room temperature for 1 hour under nitrogen. The protein was then precipitated with ice-cold acetone and purified as before. The pure tryptophan- modified protein (0.5 mg) was dissolved in 1 ml of 80% acetic acid and an aliquot after overnight hydrolysis with 6 N HCl at 105” was used to estimate the protein content of the solution by amino acid analysis with a Locarte amino acid analyzer (Locarte Company, Emperor’s Gate, London, England).

The tryptophan content of the purified labeled protein solution was determined spectrophotometrically in a Cary 15 spectro- photometer (Cary Instruments, California) with a molar ab- sorptivity of E 4000 at 365 nm (44). Owing to the presence of a pale yellow color in apoferritin (5) it was necessary to include a reagent blank by treating some of the original reduced and car- boxymethylated protein as described above in the absence of 2-nitrophenylsulfenyl chloride.

Method g--The method used was essentially that of Edelhoch (45). Horse spleen apoferritin (0.2 to 0.7 mg per ml) was allowed to unfold in the presence of 6 M guanidine hydrochloride pH 6.50 (Mann, ultra-pure) for 18 hours at 37”. Absorption spectra were again obtained with a Cary model 15 spectrophotometer.

Estimates for the tryptophan and tyrosine content were evaluated as shown below. Including the contribution of cys- teinyl residues to the absorption at 280 and 288 nm we have:

Em, = Nt, 5690 + M,,, 1280 + L,,, 120 (1)

Em = Nt, 4815 + Mt,, 385 + L,, 75 (‘4

where N, M, and L are the number of moles of tryptophan, tyrosine, and cysteine per mole of protein. Eliminating Mtyr we have, Ntrp = E&3103 - E&10318 - L&83.56. Thus by measuring the absorption at 280 and 288 nm and by using the literature value for LCyS (46), Ntrp can readily be evaluated.

Amino Acid Analysis

The amino acid analysis was carried out with a Locarte amino acid analyzer (Locarte Company, London) after 16 hours of hydrolysis with 6 N HCl at 110”. Methionine was determined after carrying out the hydrolysis in a vacuum while cysteine was determined as cysteic acid. Tryptophan was determined both by the method of Edelhoch (45) and by conversion to the 2- nitrophenylsulfenyl derivative as described.

RESULTS AND DISCUSSION

Molecular Weight of Apojerritin Subunit

SDS-Polyaerylamide Gel Electrophoresis-Fig. 1 shows exam- ples of the separation of several different polypeptide chains used as molecular weight markers in this study. When the electro- phoretic mobilities of such markers were plotted against the logarithm of molecular weight of the polypeptide chains, a rela- tionship was obtained as shown in Fig. 2. It can be seen from this that an inflection occurs when the molecular weight falls below 11,000. The existence of such an inflection was first re- ported by Dunker and Rueckert (26) and was explained as a reflection of less effective sieving of small molecules. These workers showed that for 5% polyacrylamide gels the re1ationsh.p

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of July 10, 1971 C. P. :I. Bruce and R. R. Crichton 4201

80

00

40

30

^ 20 7 z x

1 M

L ia 10

B 2 *

s

6

4

3

2

FIGI. 1. SDS-polyacrylamide gel electrophoresis. Typical gels obtained after electrophoresis; staining and destaining as de- scribed in the text are shown. A, Gel 1, contains myoglobin, chymotrypsinogen, pepsin, leucine aminopeptidase, and serum albumin, from left to right. Above is a densitometric scan of this gel. Ge2 I, horse spleen apoferritin. B, Gel 1, human transferrin showing the minor components (see text) and horse spleen apofer- ritin; Gel 2, ovalbumin and insulin; Gel 3, bovine serum albumin and insulin.

of molecular weight to electrophoretic mobilities was biphnsic. In our studies of the structure of horse spleen apoferritin we became interested in the purification and characterization of the peptides obtained from cyanogen bromide cleavage (46-48). We therefore decided to try to extend the range for molecular weight estimation by determining the mobilities of cyanogen bromide peptides of well characterized proteins (cytochrome c, lysoeyme, and myoglobin) in the hope that we could calibrate the region of steeper slope. This was performed on 10% gels as before and it was found that there was an apparent linearity in the region of 1,500 to 8,000 but the steepness of the slope made any molecular weight estimation from this region highly inaccurate.

Lysozyme and notably ribonuclease A have been described as “anomalous” proteins inasmuch as they migrate more slowly than expected. In our study and that of Weber and Osborn (25) such deviation from expected values was not found. Shapiro, Vinuela, and Maize1 (28) used 5% gels and since the “critical point” for such gels is about 20,000 (26) then this could explain the deviation for lysozyme and ribonuclease A. It is known that ribonuclease still has activity in the presence of SDS2 and this

* J. V. Maieel, personal communication.

Chpotrypincgen A

Cytoshme C, CNBr peptidell.

b C,e,sh,me C, C NBr peptide IE

I I I I I I I I I I I I 88 1 L I I

0.3 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

Electropboretie Mobthty

FIQ. 2. SDS-polyacrylamide gel electrophoresis. The electro- phoretic mobilities of the molecular weight markers used in this study are shown as a function of their respective molecular weights. The relationship so obtained appears to be biphasic and this is discussed further in the text.

suggests that ribonuclease is unusually resistant to unfolding in the presence of this detergent. Since Dunker and Rueckert (26) have shown that unfolding causes an decrease in mobility com- pared to the native protein then this could explain the anomalous behavior of this particular protein.

The average electrophoretic mobility for the dissociated pro- tein was calculated to be 0.734 i 0.004 (horse spleen apoferritin) and 0.73 f 0.01 (horse spleen ferritin). These represent the mean values and standard deviations of 15 and 4 determinations, respectively. (In the case of ferritin, a narrow band containing iron was observed to travel a short distance into the gel.) These electrophoretic mobilities correspond to molecular weights of 18,300 ZIZ 300 and 18,400 f 500, respectively.

It has been suggested that transferrin which has two separate iron-binding sites consists of two polypeptide chains (49, 50) of molecular weight about 40,000. From SDS-polyacrylamide gel electrophoresis we found that transferrin had an electro- phoretic mobility of 0.161 f 0.006 which corresponds to a molecular weight of 76,500 ZIZ 1,500. This value is in close agree- ment with that determined for the reduced and carboxymethyl- ated protein in 8 M urea (51) and also for the protein in the presence of 6 M guanidine hydrochloride (31, 32). From such molecular weight studies it seems that transferrin probably con- sists of one polypeptide chain and this view is in agreement with

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4202 Subunit Molecular Weight of Apofewitin Vol. 246, No. 13

CdOl.~~

Y-globulin, H chain

\....I Ovalbumin

Alcohol dehydmgenase Creatine phosphokinare

y-globulin, L chain

I I 1

0.05 0.1 0.15 0.20 0.25 0.30 0.35

Kd

FIG. 3. Molecular weight determination by gel filtration on Sepharose 6B in the presence of 6 M GuHCl. Distribution coeffi- cient (Kd) is shown as a function of molecular weight and the linear relationship was computed by the method of least squares analysis.

the results of Elleman and Williams (52) who identified 34 unique cysteic acid peptides in ovotransferrin (ovotransferrin contains 31 moles of half-cystine per 80,000 g of protein). On electro- phoresis of transferrin a minor band was found in all cases (Fig. 1) with an electrophoretic mobility of 0.205 fi 0.004 which corre- sponds to a molecular weight of 69,000 f 1,000. This species accounted for about 10% of the total protein and may in fact merely represent an impurity in the preparation.

Agurose Gel Filtration-The molecular weight markers used in this study were cochromatographed with blue dextran and tryptophan which gave values for the exclusion volume, V,, and internal volume, Vi, respectively. No difficulties arose from the use of molecular weight markers with elution volumes, ve, close to the void volume.

Fig. 3 shows the plot of the distribution coefficient, Kd (53), versus the logarithm of molecular weight markers in this study (54). A linear relationship can be expressed in the form:

log (molecular weight) = 5.086 - 2.631 kd

Such a linear expression was found by Davison (55) although a more recent and extensive study has suggested a sigmoidal re- lationship (23). The value estimated for the distribution coef- ficient, Kd, of dissociated horse spleen apoferritin was 0.309 st 0.003 and this represents the mean value and standard deviation

-4 2 1 I

46 47 48 49 50 51

r2 (cm’)

FIG. 4. Molecular weight estimation by sedimentation equilib- rium in the analytical ultracentrifuge. A, shows the results of a typical sedimentation equilibrium experiment performed with acetic acid-dissociated apoferritin in dilute glycine buffer (‘Ma- terials and Methods”). dlnC/dr2 for this line was calculated to be 0.4439 by the method of least squares analysis (regression coeffi- cient = 0.9981). B, shows the results of a similar sedimentation experiment with guanidine-dissociated apoferritin. dlnC/d+ in this case was computed to be 0.2774 with a regression coefficient of 0.9971. Both runs were performed at 20,000 rpm and 20’.

of eight determinations. This corresponds to a value of 18,800 i 400 for the molecular weight of the apoferritin subunit and this result is in good agreement with the value obtained by SDS- polyacrylamide gel electrophoresis. The value obtained for the distribution coefficient of transferrin (human and horse) was 0.0766 f 0.0025 which corresponds to a value of 76,600 i 1,200 obtained by extrapolating the experimentally determined rela- tionship. This value provides further confirmatory evidence for the view that transferrin consists of one polypeptide chain.

Tanford, Kawahara, and Lapanje (33) concluded from the increase in the intrinsic viscosity accompanying protein de- naturation in concentrated guanidine hydrochloride that the polypeptide chain adopts a conformation close to a true random coil and thus, since there is no shape dependence the proteins will elute as a function of the polypeptide chain length (55). Because of the fact (33) that the time required to attain complete unfold- ing is about 1 hour it was decided, as in the study by Fish et al. (23), to exclude proteolytic enzymes as molecular weight markers.

In the present study insulin was found to elute as two peaks with partition coefficients of 0.792 XI= 0.002 and 0.837 & 0.002 which presumably correspond to the B and A chains, respec- tively. However, these lower molecular weight peptides do not satisfy the linear relationship already given and no attempt has yet been made to calibrate this region of low molecular weight in the range 2,000 to 10,000 daltons.

Analytical Ultracentrifugation-Data from the sedimentation equilibrium experiments were treated according to the equation describing the concentration distribution at equilibrium (56).

2RT Mw = (1 - pe)w2

d In C .- &.!a

where % is the whole cell weight average molecular weight, w is the angular velocity of the rotor, and 8’ is the effective partial specific volume of the protein.

Plots of In C versus r2, where C represents the protein concen- tration (in milligrams per ml) and r the distance from the center of rotation, were found to be rectilinear both for the protein subunit in glycine-HCl buffer and in 6 M guanidine hydrochloride as shown in Fig. 4. Such linear relationships which imply a

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of July 10, 1971 C. F. A. Bryce and R. R. Crichton 4’203

monodisperse macromolecular species were computed by least squares analysis with a PDP-8/L computer (Digital Equipment Corporation, Reading, England).

The partial specific volume (v) for horse spleen apoferritin was calculated from the amino acid composition (Table II) by the method of Cohn and Edsall(58) and found to be 0.731 ml per g, whereas the value previously published for the pa,rtial specific volume from pycnometric estimation was 0.747 ml per g (21).

The molecular weight of apoferritin dissociated by 67% acetic acid and dialyzed into glycine buffer was 18,400 =t 800 with v = 0.731, or 19,500 f 900 with v = 0.747.

Unfortunately, the data for the guanidine hydrochloride-dis- sociated subunit was more difficult to interpret since there still seems to be some doubt at present about the effect of high guani- dine hydrochloride concentration on the partial specific volume (8) of proteins. Just as there have been several well documented reports of a 1 to 2% decrease in 7 (59-61), so too have there been several which indicate no change (62-65), or even a slight increase (66). There is also some doubt concerning the extent of preferential binding of guanidine to proteins (62, 63). That such apparent ambiguities exist does not, we feel, allow us to forejudge, but merely to express the various possibilities (Table III).

It has been shown that apoferritin polymerizes to form dimers, trimers, etc. (67). That the protein behaves as an associating- dissociating system is reflected in our preliminary attempts at an estimation of the molecular weight of the undissociated protein from sedimentation equilibrium.3 This situation introduces a degree of uncertainty and for this reason we have not as yet ob- tained an unambiguous value for the undissociated apoprotein and this area, which we feel merits more consideration, is cur- rently under active investigation. The presently accepted molecular weight of the undissociated apoferritin lies within the range 430,000 to 465,000 and this represents estimations by a variety of physical techniques (15, 21, 22).

Quantitative Tryptophan Determination-Another useful method for a molecular weight estimation is to calculate the minimum molecular weight from the amino acid composition with the amino acid which appears least frequently in the pro- tein. In horse spleen apoferritin tryptophan represents the amino acid in the smallest amount and the minimum molecular weight based on the tryptophan content determined by Harrison, Hofmann and Mainwaring (18) was calculated to be 23,000. As this value for the molecular weight supports the view of a 20- subunit structure, we decided to reinvestigate the tryptophan content of the protein in the hope of resolving the conflicting data.

It has been shown that by using 2-nitrophenylsulfenyl chloride it is possible to generate, in a tryptophan-containing protein, a chromophore, the absorption of which can readily be monitored in the visible region of the spectrum (44). It has also been shown that sulfenyl halides react with sulfhydryl groups to form mixed disulfides (68, 69). However, the reaction of the modifying agent with any cysteinyl residues in the protein can be readily reversed under mild alkaline conditions thereby allowing accur- ate tryptophan evaluations (44). The reliability and general applicability of such a procedure has been shown for several well characterized proteins (43) and has the advantage that multiple reactivity with tryptophan residues does not occur (70).

The average value obtained from eight such determinations

*R. R. Crichton, R. Eason, A. Barclay, and C. F. A. Bryce, unpublished observations.

TABLE II

Amino acid composition of horse spleen apojerritinas b

Amino acid residue No. of residues per 18,500 daltons

Cysteic acid. ........................ Aspartic acid. ....................... Threonine .......................... Serine ............................. Glutamic acid. ................ ..... Proline ............................ Glycine ........................... Alanine ............................ Valine ......................... Methionine ......... ............ Isoleucine ....................... Leucine ............................ Tyrosine .................... Phenylalanine .................. Histidine ........................ Lysine ....................... Arginine ......................... Tryptophana. ......................

2.87 17.31 5.48 8.97

23.85 2.82 9.87

13.97 (3.93 2.76 3.49

24.95 4.99 7.33 5.78 8.73 9.45 2.09

D Determined separately as described in text. b For comparison with previously published values see Refer-

ence 57.

TABLE III

Sedimentation equilibrium experiments on horse spleen apoferritin dissociated by 6 M guanidine hydrochloride

Extent of preferential binding

No binding. . . . . 0.05 g of GuHClj per g

of protein.. 0.10 g of GuHCl per g

of protein.. .

- I Partial specific volume ($1

0.747’ o.74ob --

21,400 20,100

20,300 19,100

19,300 18,100

- 0.731c / 0.724d 1 0.717’

-

18,700 17,700 16,800

17,800 16,800 16,000

16,800 15,900 15,100

a The value for % from Reference 21. b The value for B (Reference 21) with a 1% correction (61). c The value for B (Reference 21) with a 2% correction (60).

This is also the value for e calculated from amino acid composi- tion (see text).

d The value for 8 calculated from the amino acid composition with a 1% correction (61).

6 The value for B calculated from the amino acid composition with a 2% correction (60).

f GuHCl, guanidine hydrochloride.

was calculated to be 52.6 f 1.5 moles of tryptophan per mole of undissociated horse spleen apoferritin. This figure is based on a molecular weight of 480,000 daltons so as to be directly compar- able with the data of Harrison and Hofmann (17) and Harrison et al. (18) who found 21 moles by a modification of the method of Spies and Chambers (71). The value obtained from the present study would imply 2.52 f 0.07 tryptophanyl residues per subunit of molecular weight 23,000 daltons and 2.03 f 0.06 tryptophanyl residues per subunit of molecular weight 18,500 daltons.

The tryptophan content of the protein was also determined spectrophotometrically by the method of Edelhoch (45). The value obtained by this method was 55.6 f 1.0 moles of trypto- phan per mole of undissociated horse spleen apoferritin based on a molecular weight of 480,000 as before. This corresponds to a

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4204 Subunit Molecular Weight of Apojerritin Vol. 246, No. 13

TABLE IV Summary of molecular weight obtained for the polypeptide chain of

horse spleen apoferritin

Molecular weight Method of determination

18,300 f 300 SDS-polyncrylamide gel electrophoresis

18,800 f 400 G M guanidine hydrochloride gel filtration

18,400 f 800 Sedimentation equilibrium performed on pro- 19,500 f 900 tein dissociated by 67yo acetic acid and dia-

lyzed into dilute glycine buffer with B = 0.731 with B = 0.747

15,100-18,700 Sedimentation equilibrium performed on protein 16,806-21,400 dissociated by 6 M guanidine hydrochloride

(for explanation of range see Table III), with 6 = 0.731 with I = 0.747

value of 2.14 + 0.04 per 18,500. It can be seen that this value is in good agreement with that determined by chemical modifica- tion. Also by using only the literature value of the number of cysteinyl residues (46) and substituting this value into either Equation 1 or 2 then the values computed for the tyrosine con- tent are 5.08 tyrosyl residues per subunit of molecular weight 18,500 or 6.17 residues per subunit of molecular weight 22,500 which are in excellent agreement with literature values (18, 57), and this serves as a useful further check on the validity of the tryptophan determination.

Subunit Struclure of Apoferriti?z-Table IV summarizes the re- sults for the molecular weight determination of the subunit. It is clear that the polypeptide chains comprising the horse spleen apoferritin molecule have a molecular weight of the order of 18,500, and this value is further supported by the tryptophan content, described in the previous section. For the present dis- cussion, assuming the undissociated molecular weight to be around 460,000, this would imply 23 to 25 subunits per molecule of undissociated apoferritin, although a value of 24 would seem most likely (72, 73) on grounds of symmetry.

The chemical evidence on which the value of 20 subunits is based merits some comment. The observation (17, 57) that 25 peptides are liberated by the action of trypsin, and that this is in good agreement with the lysine plus arginine content of the subunit (23 per 23,000 daltons) might well be misleading. Per- haps the best illustration of this is the case of turnip yellow mosaic virus (74) in which, despite the fact that 60% of the tryptic digest remains at the origin as a ninhydrin-negative spot, the expected number of tryptic peptides, namely 11 to 12, can be visualized. However, only seven of them represent unique se- quences, the rest being derived from incomplete digestion and unexpected chymotryptic cleavages. In the present case careful determination of the number of peptide bonds cleaved during trypsin digestion of apoferritin shows quite clearly that even after 16 hours the theoretical extent of cleavage is not nearly attained.4

We have shown that the tryptophan content of apoferritin is considerably higher than was previously thought, and it is clear that determination of tryptophan by complex calorimetric meth- ods (71) can lead to inaccurate estimates of the content of this

4 It. R. Crichton, unpublished observations.

amino acid. Methods available for quantitative estimation of N-acetyl groups would appear to be less accurate than would be required to distinguish between 20 and 24 subunits (75), although a value of 19.6 f 1 has been reported (20).

The molecular weight of proteins dissociated in SDS, as de- termined by sedimentation in the ultracentrifuge, is clearly de- pendent on an accurate estimation of the amount of detergent bound to the protein. We conclude that the value determined by Hofmann and Harrison (19) was too high on account of a failure to completely remove all of the SDS. If the amount of SDS bound to the protein was underestimated, the molecular weight of the subunit would clearly be too high. If one assumes that the binding of SDS for globular proteins is 1.4 g of detergent per g of protein, as has been recently suggested by Reynolds and Tanford (76), and that the resultant v for the protein-SDS com- plex is 0.8275, the value for the subunit molecular weight of apoferritin from the data of Hofmann and Harrison (19) can be calculated as 18,100 to 19,600 which is in marked contrast) to the value of 25,000 to 27,000 found by them.

The final piece of chemical evidence advanced in favor of the idea that there are 20 subunits is the fact that carboxypeptidase B liberates 19 moles of arginine and 15 moles of lysine (20), and that the COOH-terminal octapeptide has been isolated and shown to end in a Lys-Arg sequence (20). We have not con- firmed this value and can therefore make no comment.

It has been suggested recently (77) that the subunit molecular weight is of the order of 12,000. This value was based on gel filtration on a column of Sephadex G-200 eluted with buffer con- taining 0.1% SDS, in which SDS-dissociated protein eluted “as though it had a molecular weight lower than chymotrypsinogen, but similar to cytochrome c.” However, we have never observed dissociated apoferritin subunits to chromatograph on columns or to migrate on gels in the vicinity of cytochrome c.

It is most difficult to reconcile our conclusion that there are 24 subunits in the horse spleen apoferritin molecule with the x-ray crystallographic data of Harrison (11). We can simply point out that 24 was a value originally suggested by Harrison (15) from preliminary x-ray data, but that the evidence for a 5-fold axis does seem to be quite strong. However, we must emphasize that our data tends to imply a value of 24 for the number of sub- units assuming the presently accepted values for the molecular weight of the undissociated apoprotein. These values range from 430,000 to 465,000 (15, 21, 22)s and represent the results obtained by four separate and distinct techniques. Independent chemical data obtained in this laboratory confirm that the sub- units of molecular weight 18,500 are chemically identical. There is likely to be a greater degree of uncertainty associated with the value for the undissociated protein molecular weight than with that of the subunit. That this error would amount to 20%, as would be necessary in all four cases if there were in fact 20 sub- units of molecular weight 18,500, does not seem probable.

Acknowledgments-We would like to thank Mr. A. Barclay for carrying out the ultracentrifugation runs and Dr. R Eason for advice and much appreciated criticism. Our thanks are also due Professor J. N. Davidson, F.R.S., and Professor R. M. S. Smellie for provision of facilities.

Addendum-Since the preparation of the final form of this manuscript it has been found possible to effect peptide chroma-

5 W. W. Fish and I. Bjork, personal communication.

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Issue of July 10, 1971 C. F. A. Bryce and R. R. Crichtm

tography in the molecular weight range 1400 to 8000 on the same 38. column of Sepharose 6B equilibrated with 6 M guanidine hydro- chloride. The molecular weights calculated from such studies 39. for well characterized peptides afford an accuracy of better than 10%. A description of this technique together with its applica- tion to the determination of the molecular weight of the peptides 40. obtained from the cyanogen bromide cleavage of apoferritin will be published shortly. 41.

REFERENCES 42. 1. LAUFBERCJER, V., Bull. Sot. Chim. Biol., 19, 1575 (1937). 2. MAZUR, A., AND SHORR, E., J. Biol. Chem., 182, 607 (1950). 43. 3. FARR,~NT, J. L., Biochim. Biophys. Acta, 13, 569 (1954). 4. GRAIUICK, S., Physiol. Reu., 31, 489 (1951). 44. 5. GRANICK. S.. AND MICHAELIS. L.. J. Biol. Chem., 147, 91 (1943). 6. BIELIC~, k. ‘J., AND BAYER,. E.; Natururissenschajten, 42, 466

(1955). 7. MAZUR, A., CARLETON, A., ALYD CARLSEN, A., J. Biol. Chem.,

236, 1109 (1961).

45. 46.

8. RICHTER, G.. W., J. Biophys. Biochem. Cytol., 6, 531 (1959). 9. BESSIS.M.. AND BRETON-GORIUS. J.. C. R. Hebd. Seances Acad.

10.

11. 12.

13.

Sci. Paris, 260, 1360 (1960). ’ VAN BRUGOEN, E. F. J., WIEBENGB, E. H., AND GRUBER, M.,

J. Mol. Biol., 2, 81 (1960). HARRISON, P. M., .Z. Mol. Biol., 6, 404 (1963). FISCHBACH, F. A., AND ANDEREGG, J. W., J. Mol. Biol., 14,

458 (1965). BIELIG, H. J., KRATKY, O., ROHNS, G., AND WAWRA, H., Tetra-

hedron Lett., 38, 2701 (1964). 14. BIELIG, H. J., KRATKY, O., STEINER, M., AND WAWRA, H.,

15. 16. 17.

Monafsh. Chem., 94, 989 (1963). HARRISON, P. M., J. Mol. Biol., 1, 69 (1959). HARRISON, P. M., Acta Crystallogr., 13, 1051 (1960). HARRISON. P. M.. AND HOFMANN, T., J. Mol. Biol., 4, 239

18. (1962). ’

HARRISON, P. M., HOFMANN, T., AND MAINWARING, W. I. P., J. Mol. Biol.. 4. 251 (1962). 59.

19.

20.

21. 22.

HOFMANN, T., ‘AND HARRISON, P. M., J. Mol. Biol., 6, 256 (1963). 60.

MAINWARING, W. I. P., AND HOFMANN, T., Arch. Biochem. Biophys., 126, 975 (1968). 61.

ROTHEN, A., J. Biol. Chem., 152, 679 (1944). RICHTER, G. W., AND WALICER, G. F., Biochemistry, 6, 2871 62.

(1967). 23.

24. 25. 26.

FISH, W. W., MANN, K. G., AND TANFORD, C., J. Biol. Chem., 63. 244, 4989 (1969).

MAIZEL, J. V., Science, 161, 988 (1966). 64. WEBER, K., AND OSBORN, M., J. Biol. Chem., 244, 4406 (1969). DUNKER A. K., AND RUECI~ERT, R. R., J. Biol. Chem., 244, 65. ---. ..‘..-.

47. 48.

49.

50. 51.

52.

53. 54. 55. 5ci. 57. 58.

5074 (lY6Y). SHOME, B., BROWN, D. M., HOWARD, S. M., AND PIERCE, J.

G.. Arch. Biochem. Biowhus.. 126. 456 (1968). 27. SHAPIRO, A. L., SCHARFF, M. D., MAIZEL, J. V., AND UHR, J.

W., Proc. Nat. Acad. Sci. U. S. A., 66, 216 (1966). 28. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V., Biochem.

Biophys. Res. Commun., 26, 815 (1967). 29. SHAPIRO, A. L., AND MAIZEL, J. V., Anal. Biochem., 29, 505

(1969).

66.

67.

68.

69.

GREEN, R. W., AND Mc~A;, k HI, J. kol.‘Chem., 244, 5034 (1969).

HARRISON, P. M., AND GREGORY, D. W., J. Mol. Biol., 14, 626 (1965).

SAKAICIBARA, S., AND TANI, H., Bull. Chem. Sot. Jap., 29, 85 (1956).

30. CRICHTON, R. R., AND BRYCE, C. F. A., Fed. Eur. Biochem. Sot. Lett., 6, 121 (1970).

PARKER, A. J.. AND KHARASCH, N., J. Amer. Chem. Sot., 82.

31. FISH, W. W., MANN, K. G., Cox, A. C., AND TANFORD, C., Fed. Proc., 28, 405 (1969).

70. 3071 (i960). ’

BARMAN. T. E.. AND KOSHLAND. D. E.. JR.. J. Biol. Chem.. 242, 5i71 (196j).

32. AISEN, P., KOENIG, S. H., SCHILLINGER, W. E., SCHEINBERG, I. H., MANN, K. G., AND FISH, W., Nature, 226,859 (1970).

33. TANFORD, C., KAWAH.~RA, K., AND LAPANJE, S., J. Amer. Chem. Sot., 89, 729 (1967).

71. HARRISON, P. M., AND HOFMANN, T., Biochem. J., 80, 38P (1961).

72. CRICK, F. H. C., AND WATSON, J. I)., Nature, 177, 473 (1956). 73. CASPAR, D. L. D., AND KLUG, A., Cold Spring Harbor Symp.

34. SUND, H., WEBER, K., AND MOLBERT, E., Eur. J. Biochem., 1, &ant. Biol., 27, 1 (1967). -. 40 (1967). 74. HARRIS, J. I., AND HINDLEY, J., J. Mol. Biol., 13, 894 (1965).

35. CASTELLINO, F. J., .~ND BARICER, R., Biochemistry, 7, 2207 75. DE GROOT, K.,,HOENDERS, H. J., GERDING, J. J. T., AND (1968). BLOEMENDAL, H., Biochim. Biophys. Acta, 207, 202 (1970).

36. JORNVALL, H., AND HARRIS, J. I., Abstracts of the Federation of 76. REYNOLDS, J. A., AND TANFORD, C., J. Biol. Chem., 246, 5161 European Biochemical Sciences, Praha, 1968, abstract 759. (1970).

37. DAWSON, I>. M., EPPENBERGER, H. M., AND KAPLAN, N. O., 77. SMITH-JOHANNSON, H., AND DRYSDALE, J. W., Biochim. Bio- J. Biol. Chem., 242, 210 (1967). phys. Acta, 194, 43 (1969).

BOVEY, F. A., AND YANARI, S. S., in P. D. BOYER, H. LARDY, AND K. MYRBACK (Editors), The enzymes, Vol. IV, Academic Press, New York, 1960, p. 63.

BARGETZI, J. P., SAMPATH KUMAR, K. S. V., Cox, D. J., WALSH, K. A., AND NEURATH, H., Biochemistry, 2, 1468 (1963).

DAYHOFF, M. O., AND ECK, R. V., Atlas of protein sequence and structure, National Biomedical Research Found&on, Sil- ver Spring. Maryland. 1967-1968.

SCHACHMANY’H. K., AND EDELSTEIN, S. J.. Biochemistry, 6, 2681 (1966).

CANFIELD, R. E., AND ANFINSEN, C. B.. J. Biol. Chem.. 238. 2684 (1963). ’

I _

BOCCU, E., VERONESE, F. M., FONTANA, A., AND BENASSI, C. A., Eur. J. Biochem.. 13. 188 (1970).

SCOFFONE, E., FONTANA, k., END ROCH;, R., Biochemistry, 7, 971, 980 (1968).

EDELHOCH, H., Biochemistry, 6, 1948 (1967). CRICHTON, R. R., AND BARBIROLI, V., Fed. Eur. Biochem. Sot.

Lett., 6, 134 (1970). CRICHTON, R. R., Biochem. J., 117, 34P (1970). CRICHTON, R. R., 8th International Technicon Symposium on

Automaiion in Analytical Chemistry, London, 1969, in press. BAKER, E., SHAW, D. C., AND MORGAN, E. H., Biochemistry,

7, 1371 (1968). JEPPSSON, J. O., Acta Chem. &and., 21, 1686 (1967). GREENE, F. C., AND FEENEY, R. E., Biochemistry, 7, 1366

(1968). ELLEMAN, T. C., AND WILLIAMS, J., Biochem. J., 116, 515

(1970). GELOTTE, B. J., J. Chromatogr., 3, 330 (1960). PORATH, J., Pure Appl. Chem., 6, 233 (1963). DAVISON, P. F., Science, 161, 906 (1968). YPHANTIS, D. A., Biochemistry, 3, 297 (1964). CRICHTON, R. R., Biochim. Biophys. Acta, 194, 34 (1969). COHN, E. J., AND EDSALL, J., Proteins, amino acids and pep-

tides, Reinhold Publishing CorDoration. New York. 1943. p. 3?0.

- * I ,

MARLER, E., NELSON, C. A., AND TANFORD, C., Biochemistry, 3. 279 (1964).

SMALL, P. A.; AND LAMM, M. E., Biochemistry, 6, 259, 267 (1966).

KIELLEY, W. W., AND HARRINQTON, W. F., Biochim. Biophys. Acta, 41, 401 (1960).

ULLMAN, A., GOLDBERG., M. E., PERRIN, D., AND MONOD, J., Biochemistry, 7, 261 (1968).

REITHEL, J. F., AND SAKURA, J. D., J. Phys. Chem., 67, 2497 (1963).

YUE, R. H., PALMIERI, R. H., OLSON, D. E., AND KUBY, S. A., Biochemistry, 6, 3206 (1967).

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. F. A. Bryce and R. R. CrichtonWEIGHT OF THE SUBUNIT

The Subunit Structure of Horse Spleen Apoferritin: I. THE MOLECULAR

1971, 246:4198-4205.J. Biol. Chem. 

  http://www.jbc.org/content/246/13/4198Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/246/13/4198.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from