Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 210, No. 2, September, pp. 748-761, 1981

Subunit Composition of a High Molecular Weight Oligomer:

Limulus polyphemus Hemocyanin

MICHAEL BRENOWITZ,* CELIA BONAVENTURA,* JOSEPH BONAVENTURA,*r’ AND ELISABETTA GIANAZZAt

*Department of Baochemastry and Manne Baomedzcal Center, Duke Unaversaty Marane Laboratory, Beau&t, North Carolina 28516, and TDepartment of Baochemastry, Universaty of Malano,

Vaa Celorza i, Malano 20133, Italy

Received January 26, 1981

The hemocyanin of Lamulus polyphemus is a Is-subunit aggregate. This 3.3 X 106-dalton

oligomer is composed of structurally and functionally heterogeneous subunits. Using

polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp.

PhysaoZ. Ser. B 130.28.S292,19’79) observed 12 bands; while using immunoelectrophoresis,

M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physaol. Baochem. 87,41’7- 418,1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C Bonaventura, and M Bren-

owitz. (Arch. Baochem. Baophys. 196,324-339,1979) observed 8 subunits. To proceed with

an analysis of subunit roles in assembly it is first necessary to determine the number

of distinct subunits Refinement of the chromatographic separation procedures has led

to the isolation of 8 immunologically distinct subunits as well as additional charge isomers

which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15

bands and isoelectric focusing up to 17. On the basis of extensive control experiments,

including composit acrylamide-agarose immunoelectrophoresis and checks for confor-

mational isomers, aggregation, proteolysis, and other types of degradation, we conclude

that the electrophoretic heterogeneity of immunologically identical subunits is not ar-

tifactual. We have extended the nomenclature used by Lamy et al. (1979) to mclude the

electrophoretm heterogeneity by using primes (‘) to denote electrophoretically distin-

guishable subunits which are immunologically identical A number of patterns have

become apparent by correlating the results obtained by the different techniques For

example, immunologically pure subunit II, which shows 3 bands on alkaline electropho-

resis, IS in fact a mixture of electrophoretically distinct subumts II, II’, II”. Except for

subunits II, II’, and II” immunoelectrophoretically identical subunits are typically ho-

mogeneous on sodium dodecyl sulfate-gels However, slight differences m the apparent

molecular weight are observed on high-resolution gels between immunologically unre-

lated subunits. The immunological identity and electrophoretic differences suggest that

the charge isomers which are immunologically identical have similar antigemc surfaces.

If a charge substitution IS not in a critical location, we would expect the electrophoretically

distmct but immunologically identical subunits to have identical assembly roles. Com-

parison of the results for Lamulus hemocyanin with the hemocyanm of related species

Eurypelma calZfornzcum and Androctanus australts, which have 7 and 8 immunologically

distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24

to 48 subunits of Lzmulus does not require more extensive subunit complexity

Hemocyanins are the copper-containing respiratory proteins of a variety of species of arthropods and molluscs. The hemocyanins typically have multiple subunits, high molecular weights, and oxygen binding that is modulated by pH and ions. The

partial isolation and characterization of

i To whom reprint requests and correspondence

should be directed

the constituent polypeptides of Limulus hemocyanin were a clear demonstration of structural and functional heterogeneity in an arthropod hemocyanin (l-3). The presence of cooperative oxygen binding and allosteric interactions in the native molecules of Limulus hemocyanin implies that the single oxygen-binding site of each subunit is influenced by the conformation of the surrounding polypeptide chain. In

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748

SUBUNIT HETEROGENEITY OF Lzmulus HEMOCYANIN 749

a sense the active site is a sensitive re- porter group of the conformation of an individual subunit. The present detailed study of subunit heterogeneity is based on the concept that structurally diverse subunits can be used as probes of the confor- mational changes and protein-protein interactions involved in self-assembly and in allosteric modulation of oxygen affinity.

The demonstration of chain heterogeneity in many species of both arthropods and molluscs has established heterogeneity as the rule rather than the exception (4, 5). A systematic study of electropho- retie heterogeneity of arthropod hemocyanins has been published (6). A total of 12 bands was found for Limulus hemocyanin, an increase from the 8 bands previously identified (1, 2). Other arthropod hemocyanins analyzed showed fewer bands. Among the arthropods only the horseshoe crabs (Limulus and Tachypleus) are known to form 4%subunit aggregates (7). Mark1 et al. (6) proposed that the complexity of subunit heterogeneity was related to the ability of Limulus hemocyanin to reach this high aggregation state. However, analysis of the Limulus hemocyanin sys- tem by immunological techniques revealed only eight immunologically distinct components, some of which exhibited partial identity with other components (8,9). This difference in the apparent number of subunits indicated that no generalizations could be drawn regarding heterogeneity and the specific roles of subunits in assembly of the native molecule without a more precise study of the observed immunological and electrophoretic differences between the Limulus subunits. The correct determination of the subunit composition of a protein is a prerequisite for properly interpreting data on the structural and functional properties of the oligomeric form.

To accomplish this study, we have purified all of the immunologically distinct components observed by Lamy et al. (8) and Hoylaerts et al. (9) and some of the electrophoretically distinct components observed by Mark1 et a2. (6). By simulta- neously applying techniques of polyacryl-

amide gel electrophoresis, immunoelectrophoresis, and isoelectric focusing we have been able to consider why different num- bers of subunits have been reported.

MATERIALS AND METHODS

I. Pumjkatwm of whole hemocyanin. Adult Lim- ulus, collected around Beaufort, North Carolina, were bled by inserting a sterile needle between the cara- pace and tail and gently drawing the hemolymph into a syringe. The hemolymph was then centrifuged for 10 min at 17,000 rpm to remove the fibrous clot, and the supernatant was pelleted by centrifugation for 2 h at 106,OOOg at 4°C. The pellet was resuspended in 0.05 M Tris/HCl, with 10 mM CaCla, pH 7.0, centrifuged again, and the second pellet dissolved in the desired buffer. An alternative procedure, which re- moves the amoebocytes present in the hemolymph without lysis, was to draw hemolymph into a sterile syringe containing an equal volume of 10 mM the- ophylline, 0.05 M saline The diluted hemolymph was transferred to sterile centrifuge tubes. The unlysed cells were pelleted at 1000 rpm for 3 min. The supernatant was decanted and ultracentrifuged as above. The hemocyanin solutions used for structural analysis were made 0.02% in sodium azide and 5 mg/ ml in PMSF* by addition of concentrated stock solutions to the crude hemolymph and subsequent buff- ers. PMSF solutions were prepared immediately be- fore use.

II Subunzt pumjication. Optimum separation was accomplished with a 4 X 50-cm column with DEAE- Sephacel equilibrated in 0.05 M Tris/HCl, 10 mM EDTA, pH 89 (“stripping buffer”). A maximum of 2.7 g of “stripped hemocyanin” (purified hemocyanin dialyzed versus the “stripping buffer” overnight) was applied to the column and eluted with a B-liter 0.17- 0.4 M NaCl gradient. A flow rate of no greater than 35 ml/h was maintained, and 25-ml fractions were collected. Following elution of Zone Va the gradient was stopped and the column flushed with 0.5 M NaCl to concentrate the elongated tail of Zone Vb

To further separate the subunits present in Zone II, the fractions under the zone were pooled and rechromatographed on a 2.5 X 50-cm column of DEAE- Sephacryl equilibrated with 0.05 M Tris/HCl, 10 mM EDTA at pH 8.0. Elution was with a 2.0-liter gradient from 0.1 to 0.3 M NaCl at pH 8.0 Flow rates of 20 ml/ h or lower were necessary for optimum separation Immunologically pure subunits IIIa and IIIb were prepared by rechromatographing Zone III in the

* Abbreviations used: PMSF, phenylmethylsulfo- nyl fluoride, SDS, sodium dodecyl sulfate; DTT, di- thiothreitol, Hc, hemocyanin; tlg, thin-layer gel fil- tration

750 BRENOWITZ ET AL.

same manner as for Zone II except that a 0.12-0.33 M NaCl gradient was used.

Absorbance at 340 nm was monitored and fused- rocket immunoelectrophoresis or alkaline electrophoresis was performed to locate the distinct components from heterogeneous peaks. Pools of fractions were concentrated by vacuum dialysis and dialyzed exhaustively against stripping buffer. Purity of the subunit preparations was checked by crossed immunoelectrophoresis and alkaline electrophoresls.

III. Natzve alkalzne electrophoresis. The method of Davis (10) was used. Vertical slab gels of 18 cm length and 10% acrylamide were optimal. Gels were stained with 0 1% Coomassie blue G-250 in 25% trichloro- acetic acid and destained with l-methanol-l&water- 12-glacial acetic acid (volume ratios).

IV. SDS-electrophoreszs. Electrophoresis in SDS was performed on 6%, 18-cm vertical slab gels es- sentially by the method of Laemmli (11). Transferrin (77,000), bovine serum albumin (66,000), ovalbumin (43,000), and a-chymotrypsinogen (23,000) were used as standards The estimated molecular weights presented are the averaged values of two runs. Gels were stained with a 1% Coomassle blue solution and destained as above

V, Zmmunoelectrophoreszs. The methods used in this study are from Weeke et al (12) and are also described by Lamy et al. (8) using antiserum prepared against whole stripped km&us hemocyanins. Com- posite alkaline gel-crossed immunoelectrophoresis was performed as a standard crossed immunoelectrophoresis except that for the first dimension an alkaline acrylamide gel was run, and the appropriate strip was cut from the gel and then transferred to the second-dimension immunology plate in preparation for pouring the antibody-containing agarose Care was taken to insure uniform contact between the agarose and the acrylamide

VZ. Zsoelectmc focusing. Initial isoelectric focusing experiments on the chromatographic zones were performed on analytical thin-layer polyacrylamide gels with a total acrylamide concentration of 5% and a relative crosslinking concentration of 4% Isoelectric focusing of the pure subunits was performed on 1% Marine Collolds Isogel agarose plates as described by the firm. For both sets of experiments, 2.5% LKB Ampholines, pH range 5-7, were used. About 10 pg of the sample was applied to pieces of Whatmann 3MM filter paper which had been placed on the gel. The anolyte was 1 M phosphoric acid and the cath- olyte was 1 M sodium hydroxide Power was maintained for 90 min for the agarose plates and 5 h for the acrylamlde gels. Gels were stained with Coom- ass+le blue (13).

VZZ Ultracentwjkgatzon. Sedimentation velocity experiments were performed with a Beckman Model E analytical ultracentrifuge with Schlieren optics. Protein concentrations of about 5 mg/ml were used

unless otherwise noted. Plates were measured with a Nikon Microcomparator. Sedimentation coeffi- cients were calculated from the movement of the maximum ordinate of the Schlieren peak and were corrected to .!&w by standard procedures.

VZZZ. Than-layer gel jiltratwn. Thin-layer gel fil- tration was done on Zone V proteins on Sephadex G- 150 Superfine equilibrated in stripping buffer ac- cording to the method of Pharmacia Fine Chemicals using an apparatus from the firm. A 40-cm plate was used at an angle of 10” for 8 h. Protein concentrations were 20 mg/ml.

RESULTS

Purification and Electrophwesis

In this paper the term “Zone” will refer to a particular chromatographic peak, which may contain more than one protein. A subunit refers to one polypeptide chain which is the smallest piece obtainable without breaking covalent bonds. The terms “immunoelectrophoretically identified subunit” and “electrophoretically identified subunit” refer to the method used in identification of the subunits. In

FIG 1. Elution profiles of Hc subunits from DEAE chromatographic columns. Graph A represents a typ- ical elution of whole stripped Hc eluted from the pH 8.9 column. Graphs B and C show the rechromatography of Zones II and III, respectively. The Zones designated in Graph A are those described by Sul- livan et a2. (l), and Lamy et al. (8). The solid bars under the peaks indicate which fractions were pooled to obtain pure subunits. The striped area under Zones II and III of Fig. A are the fractions used for rechromatography.

SUBUNIT HETEROGENEITY OF Limulus HEMOCYANIN 751

FIG. 2. (a) Native alkaline gel electrophoresis at pH 8.9 of immunoelectrophoretically pure subunits (10 fig protein on each column except E, F). Sample (A) I; (B) Zone I shoulder peak; (C) Ha; (D) II; (E) II’ (5X concentrated); (F) II” (5X concentrated); (G) IIIb; (H) IIIb (shoulder peak); (I) IIIa; (J) IIIa (trailing edge); (K) IV, (L) Zone Va, subunit V; (M) intermediate pool between Va and Vb; (N) Zone Vb tailing edge, subunit (V’-VI); (0) mixture of Va and Vb as described under Pro- teolysis Artifacts; (P) whole stripped Hc. (b) Native alkaline gel of whole stripped Hc with bands indentified by direct comparison with purified subunits.

some cases a subunit that is pure immunoelectrophoretically is heterogeneous on polyacrylamide electrophoresis. The elution profile of the chromatographic procedures which permit purification of all the immunological classes of subunits and all but one of the electrophoretically dis- tinguishable ones is shown in Fig. 1. The sharp peak at the end of Zone Vb as seen in Fig. 1A is due to the 0.5 M NaCl buffer used following gradient elution of Va and represents only a concentration of the elongated tail of Zone Vb. Purification of subunits IIa, IIIa, and IIIb required re- chromatographies as shown in Figs. 1B and C. The shaded regions beneath peaks II and III of Fig. 1A indicate which fractions were pooled for further purification. Figures 2 and 3 show regular and SDS- gels, respectively, for fractions pooled as shown in Fig. 1.

The purified components were all subjected to alkaline electrophoresis, SDS- electrophoresis, immunoelectrophoresis, and isoelectric focusing. The isoelectric focusing experiment shown in Fig. 4 was performed on samples prior to rechro- matographies of Zones II and III as described in this paper. Isoelectric band identifications were made by a second series of focusing gels on more highly purified subunits (data not shown).

Plots of relative mobility versus acrylamide concentration for the alkaline electrophoresis gels yield parallel lines for the

observed bands of chromatographic Zones I-IV (data not shown). This indicates the bands are charge isomers of the same molecular weight (14). Thin-layer gel fil- tration studies previously published show that the subunits are monomers in stripping buffer, except for Zone Vb (8). Thus, the bands observed on polyacrylamide gels are not dimerization artifacts.

Polymorphic forms of the hemocyanin are also not the cause of heterogeneity. Studies of individuals of different size and sex in varied seasons, collected from both Beaufort, North Carolina, and Woods Hole, Massachusetts, revealed identical polyacrylamide and immunoelectrophoretic subunit patterns (data not shown).

- iSCOE p 0 Ii I 1 K I a

FIG. 3. Electrophoresis of immunoelectrophoreti- tally pure subunits in SDS. (A) I; (B) I, shoulder peak, (C) Ha; (D) II; (E) II’; (F) II”; (G) IIIa, (H) IIIb; (I) IIIb (shoulder peak); (J) IV; (K) V; (L) V and V’; (M)

(V-VI)

752 BRENOWITZ ET AL

- A B c DE FG

FIG 4. Isoelectric focusing gel of chromatographic zones (a) Zone 1; (b) Zone II (second column is a twofold dilution), (c)Zone III (both columns are identical samples), (d) Zone IV, (e) Zone Va, (f) Zone Vb; (g) whole stripped Hc

Structural Analysis of Zone I: Components I, F, and T’

Chromatographic Zone I, which corresponds to pure immunoelectrophoretically identified subunit I, shows two electrophoretically distinct bands on polyacrylamide gels designated I and I’ in Fig. 2a. The incompletely resolved peak on the shoulder of Zone I (Fig. 1A) is also pure immunoelectrophoretically identified subunit I and has a similar electrophoretic composition when compared to the major Zone I peak, except that band I’ is greater in quantity than I and an additional band,

I”, was present on polyacrylamide gels. Isoelectric focusing more clearly reveals the three bands of Zone I with PI’S between 6.1 and 6.3 as can be seen in Fig. 4. Subsequently, experiments with immunologically and electrophoretically pure subunits allowed assignment of the specific bands. SDS-electrophoresis (Fig. 3, columns A and B) reveals a single band with an apparent molecular weight of 68,200 for subunit I with samples taken from both the main peak and the shoulder peak.

Structural Analysis of Zone II: Components II, II’, and II”, and Component Ila

The refined chromatographic techniques used in this study resolved Zone II into two separate peaks (Fig. 1B). Crossed-line immunoelectrophoresis of samples from the three partially resolved peaks of the leading peak shows that immunoelectrophoretically identified component II is the major protein present (data not shown). Contamination by IIa is less than 1% as determined by quantitative crossed immunoelectrophoresis. Polyacrylamide gels show that each of the partially resolved peaks contains an electrophoretically distinct band of high purity, designated as

t

FIG. 5 Crossed-line immunoelectrophoresis with whole stripped Hc in the sample well and immunologically pure IIa (a), IIIa (b), and IIIb (c) in the reference line (d) Crossed immunoelectrophoresis of an equimolar mixture of purified subunits II, II’, and II”; (e) as (d) except equimolar mixtures of subunits II, II”. (f) Crossed immunoelectrophoresis of subunit II in the sample well and subunit II” in the reference line. All experiments were performed using antibody against whole stripped hemocyanin.

SUBUNIT HETEROGENEITY OF Limuhs HEMOCYANIN 753

IV I I lb

FIG. 6. Crossed immunoelectrophoresis of whole stripped Hc versus antibody against whole stripped HC

II, II’, and II”, respectively, with increas- ing electrophoretic mobility (Fig. 2a, columns D, E, and F). Columns E and F of Fig. 2a are overloaded to show the absence of II, and II’, respectively, and the very minor contamination by subunit IIa. Iso- electric focusing of purified II, II’, and II” revealed a total of four bands. The positions of these bands are indicated on the focusing pattern of a Zone II preparation, which contains II, II’, II”, IIa, and IIa’ (Fig. 4, column B). While II” focuses as the most acidic band, the two cathodal bands of the group comprise II. Band II’ is a single band slightly less acidic than II”. The pi’s range from 5.9 to 6.1. The faint band above II in Fig. 4 is contaminant I’ which was not present in more highly purified preparations. SDS-electrophoresis repro- ducibly reveals a single band for each subunit, differing slightly in mobility, which corresponds to molecular weights of 69,500, 67,500, 65,000 for II, II’, and II”, respectively (Fig. 3, columns D, E, and F).

As mentioned, the major bands of II, II’, and II”, while differing in electrophoretic mobility, are all immunoelectrophoreti- tally pure subunit II. As work on the crys- tal structure and sequence of Zone II is currently in progress this apparent heterogeneity was carefully investigated (15,

16). The immunoelectrophoretic technique, which employs electrophoresis in agarose in the first dimension, does not have the resolving power of the acrylamide alkaline slab gel. To test if any sub- tle differences could be detected between the three components the following series of experiments was performed. First II and II” were mixed in the sample well (Fig. 5e). A single asymmetric peak, with the small peak characteristic of a IIa contaminant, is observed indicating complete immunological identity of the two proteins which have slight electrophoretic differences (Fig. 5e). The same experiment with II, II’, II” mixed together showed the single symmetrical peak characteristic of the unfractionated Zone II (Fig. 5d). A crossed- line experiment between II and II” also shows total identity of the two proteins as evidenced by the complete fusion of the line with the peak (Fig. 5f). This complete identity became apparent only after control experiments, described by Negassi et al. (17), were performed to identify “false spurs” caused by the differing electropho- retie mobility of the proteins (data not shown).

The optical spectra of the three Zone II components were indistinguishable between 260 and 360 nm. The AzsO/AaO ratios calculated for II, II’, and II” are 2.92, 2.94, 3.07, respectively. Thus each subunit has the same amount of bound copper.

Immunoelectrophoretically identified component IIa, discovered by Lamy et al. (8) as a contaminant of chromatographic Zone II, was shown to have a unique immunoelectrophoretic identity. The purified protein, which was isolated from the second peak of the rechromatography (Fig. lB), was identified by crossed immunoelectrophoresis (Fig. 5a). It migrates as two closely spaced bands on polyacrylamide electrophoresis which overlap those of component I’ (Fig. 2a, column C). Two bands with PI’S around pH 5.7 were also observed by isoelectric focusing. On SDS- gels a single band of apparent molecular weight of 67,000 is observed (Fig. 3, column C). The Azm/AMO ratio calculated for IIa is identical to that for subunits II, II’, and II”.


FIG. ‘7. Crossed immunoelectrophoresis of (a) whole stripped Hc “aged” at 4°C for 7 days; (b) preparation as in (a) with 10 mM hydrogen peroxide added just prior to electrophoresis; (c) fresh whole stripped hemocyanin to which 10 mM DTT was added just prior to electrophoresis.

Structural Analysis of Zone III: Component IIIa and Components III& and IIIb

Zone III was rechromatographed as shown in Fig. 1C. Components IIIa and IIIb were identified by crossed-line immunoelectrophoresis as shown in Figs. 5b and c. Subunit IIIb migrates more rapidly than IIIa on the immunoelectrophoretic gel (Fig. 6); however, IIIb migrates more slowly than IIIa on the alkaline gel (Fig. 2a, columns G-J). IIIa has a greater molecular weight, as measured by SDS-electrophoresis (71,000), than IIIb (65,000) (Fig. 3, columns G and H). It is interesting to note that IIa, which is immunologically related but deficient relative to IIIa (8), has a lower apparent molecular weight. The IIIb peak of Fig. 1C has a partially resolved peak in its edge. A second band, designated IIIb’, is observed along with IIIb on polyacrylamide gels. Components IIIb and IIb’ each correspond to a single band, at about pH 6, upon isoelectric focusing; and subunit IIIa appears as a doublet whose pI centered at pH 5.4 (Fig. 4, column C).

Electrophoretic changes referred to as “aging” were reported for subunit IIIa by Lamy et al. (8) (Fig. ?‘a). Further inquiry has revealed that addition of DTT, a disulfide reducing agent, will shift the peak of IIIa on immunoelectrophoresis in the cathodal direction, mimicking the “aging” process (Fig. 7~). Oxidation by ferricya- nide or peroxide regenerates the original

peak (Fig. ‘7b). Analysis by polyacrylamide gels revealed similar shifts in mobility. No new bands were observed.

Structural Analysis of Zone IV: Component IV

Characterization of Zone IV has proved to be the most straightforward of all the zones. Zone IV consists of pure immunoelectrophoretically identified subunit IV. It gives evidence of homogeneity even on high-resolution alkaline electrophoresis (Fig. 2, column K). Isoelectric focusing also yields a single band with a pI of pH 6.1 (Fig. 4, column D), and SDS-electrophoresis gives a single band corresponding to a molecular weight of about 65,000 (Fig. 3, column J).

Structural Analysis of Zone Va and Zone Vb: Components V, P and VI

The immunoelectrophoretically identified component V is the sole protein in the leading part of peak Va (Fig. 1A). It migrates as a single band both on alkaline electrophoresis and on SDS-gels, having an apparent molecular weight of 65,000 daltons (Fig. 2a, column B; Fig. 3, column K). A pool of fractions taken from the re- gion between peaks Va and Vb of Fig. 1A shows two electrophoretic bands. One of these comigrates with V and another, designated V’, migrates slightly faster. (The additional anodal band in column M of Fig. 2a was shown by immunoelectrophoresis to be a contaminant identical to compo-


TABLE I

SELF-ASSOCIATION OF ZONES Va AND Vb PROTEINS

Sb s7.w

Ultracentrifuge Whole stripped He at pH 9.0 Subunit V at

pH 9.0 pH 8.0 pH ‘I.0

tk

5.0

5.0 6.89 6.45

Zone Vb (leading edge). pH 9.0 5. 7 (1:l ratio) Zone Vb (trailing edge), pH 9.0 7

Whole stripped He at pH 9.0 5

‘Buffer: I - 0.1 Tris/HCl with 10 m*r EDTA. ‘S values correspond to the relative mobility of marker proteins

whose sedimentation coefficient had previously been determined in the analytical ultracentrifuge under these experimental conditions.

nent IV.) The SDS pattern of the pool of fractions between Va and Vb is identical to that seen for component V, that is, a single band. Crossed line immunoelectrophoresis shows that V’ is identical to pure V isolated from the leading edge of Zone Va (data not shown). In isoelectric focusing components V and V’ each focus as a single band with pi’s at about pH 5.9 (Fig. 4, column E). The additional more alkaline bands shown in column E of Fig. 4 were never observed in subsequent, fresher preparations and are believed to be artifacts.

Alkaline electrophoresis of Zone Vb is characterized by a doublet, the slower band has the same mobility as the second band of the intermediate pool (V’). The additional, faster moving band is designated VI (Fig. 2a, column N) to correspond to the immunological identification described previously (8), which has shown that two immunoelectrophoretically distinct proteins are in Zone Vb. A diffuse, slowly migrating band is also observed in column N of Fig. 2a. This is believed to be dimeric material which dissociates into the two distinct monomeric bands under the influence of an electric field. The no- tation (V’-VI) will be used to describe this dimer. Attempts to clear up the banding pattern by urea-induced dissociation were not successful: The electrophoretic bands smeared badly in the presence of urea. Oxidation or reduction of the sample does not dissociate the dimer. When the (VI-

VI) dimer, isolated from the tail of Zone Vb, is subjected to SDS-electrophoresis, two bands appear: the 65,000-dalton band observed for component V (or V’) and a second band of 59,300 daltons (Fig. 3, column M). When SDS-electrophoresis is carried out in the absence of reductant the results are identical (data not shown), rul- ing out the possibility that the dimer is stabilized by disulfide bonds. The material gives 15 bands on isoelectric focusing gels spanning a pH range of 5.8 to 6.1 (Fig. 4F). Identification of these bands was not at- tempted.

Experiments using thin-layer gel filtra- tion (tlg) were performed to clarify the relationship between V, V’, and VI. The results are tabulated in Table I. Pooled fractions containing subunits V and V’ migrate as monomers. Pooled fractions containing components V’ and VI migrate as a mixture of dimers and monomers at the leading edge of Zone Vb, and predom- inantly as dimers at the trailing edge of Zone Vb. Thus, the dimer does not appear

FIG. 8. (A) Alkaline gel electrophoresis, pH 8.9: (A) fresh whole stripped Hc; (B) Zones I-IV (purified as described in section III); (C) fresh whole stripped Hc; (D) fresh mixture of Zones Va and Vb (see section III); (E) 2-week-old mixtures of Zones Va and Vb; (F) aged at room temperature, whole stripped Hc; (G) frozen and thawed, whole stripped Hc. (B) SDS electrophoresis: (A) whole stripped Hc; (B) Zones I- IV (see section III); (C) fresh hemolymph; (D) fresh Va and Vb (see section III); (E) “aged” Va and Vb; (F) whole stripped Hc; (G) aged at room temperature, whole stripped Hc, 4X concentration in (F); (H) frozen and thawed, whole stripped Hc; (I) room temperature, whole stripped Hc; (J) frozen and thawed, whole stripped Hc; (K, L) duplicates of I and J, respectively.


FIG. 9. Composite crossed immunoelectrophoresis (a) whole stripped Hc; (b) mixture of II; II’, II”, (c) mixture of Zones Va and Vb; (d) subunit II’ alone with Va added for reference. Antibody against whole stripped Hc.

to be in equilibrium with its constituent monomer. However, no dimer is observed when fresh unfractionated hemocyanin is stripped and then analyzed by gel filtra- tion, even at the high protein concentrations used with tlg.

Pure subunit V can form homodimers. The pH dependence of the monomer-dimer equilibrium of component V was stud- ied in the analytical ultracentrifuge. The monomer is favored at high pH and the transition to dimers occurs at about pH 8.0 as shown in Table I. This and the fore- going results indicate that subunit V is not a dissociation product of (V-VI) but can form a unique homodimer (V-V).

Proteolysis Artifacts A concern that surfaces frequently in

the hemocyanin literature is whether much of the observed heterogeneity is due to proteolysis or degradation. The major component in the hemolymph, other than the hemocyanin, are amoebocytes, which can be removed without lysis as described under Materials and Methods. Subsequent preparative ultracentrifugation allows for the purification of the whole hemocyanin from the hemolymph within 2 h. This material can then be stripped by dilution and immediately electrophoresed on alkaline and SDS-gels. Alternatively, fresh he-

molymph can be pipetted directly into boiling SDS buffer. In both cases the observed electrophoretic patterns are the same for these special preparations as for material prepared by the usual procedures. These results are shown in Figs. 8A, column A (alkaline electrophoresis) and 8B, columns A, C (SDS-electrophoresis). A “worst case” analysis was also performed by allowing one sample of whole unpr$ed hemolymph to sit at room temperature in an open beaker for 10 days. A second sample was frozen and thawed daily for the same period. When analyzed as above, no new bands were observed for either preparation (Fig. 8A, columns F, G, and Fig. 8B, columns G-J). Overloading the SDS-gels resulted in the appearance of traces of low-molecular-weight bands for all preparations (Fig. 8B, columns C, F, G, W.

The overlap of Zone II proteins with Zone Va and Vb proteins prevents direct identification of bands in alkaline gel electrophoresis of whole stripped hemocyanin (Fig. 2b). Since the Zone Va-Vb proteins apparently play an important and specific role in aggregation (3), a method was de- veloped to rapidly separate the Zone Va- Vb proteins from those of other zones. Whole stripped hemocyanin was applied to a 4 X 25-cm DEAE-Sephacel column


equilibrated with stripping buffer. Elution of Zones I-IV was readily accomplished with an eluant of stripping buffer made 0.2’7 M in NaCl. The bands II, II’, and II” are evident in this mixture (Fig. 8A, column B). Zones Va and Vb were subsequently eluted together with an eluant of stripping buffer made 0.5 M in NaCl. The bands corresponding to V, V’, VI, and the (V’-VI) dimer are all present when the eluted mixture is analyzed on alkaline gels (Fig. 8A, columns D and E, and Fig. 2a, column 0). On SDS-gels both the high- and the low-molecular-weight bands of (V’-VI) are observed (Fig. 8B and columns D and E). Thus, all purified components can be identified in banding patterns of the whole stripped hemocyanin as assayed by alkaline and SDS-gels and gel electrophoresis (Fig. 2b and 8B) and in immunoelectrophoresis (Figure 6).

Composite Immunoelectrophoresis A number of experiments were con-

ducted to test the possibility that diffusion was obscuring some immunological differences. The first dimension of the immunoelectrophoresis experiments is typically run in 1% agarose, where some diffusion- linked problems could occur. Problems were suspected in relation to the polyacrylamide electrophoretically distinct proteins, II, II’, II”, which are apparently immunologically identical. Tests were run in which the first dimension of a series of crossed-line immunoelectrophoresis experiments was run on polyacrylamide alkaline gels. In the second dimension the proteins are directly electrophoresed from the acrylamide into the antibody-containing agarose. Figure 9a shows the results from subjecting whole stripped Limulus hemocyanin to this procedure. Peaks were identified by electrophoretic mobility and crossed-line immunoelectrophoresis (data not shown). When this technique is used, the same precipitation peaks are observed as when 1% agarose is used in the first dimension, with the exceptions that the proteins compris- ing Zones Va and Vb are characterized by a completely fusing double peak. When a mixture of Zones Va and Vb was analyzed by this procedure, the same double pre-

cipitate was observed (Fig. 9c). The con- clusions of immunological identity which were drawn from regular immunoelectrophoresis are unaltered by the use of high- resolution acrylamide gels in the first dimension. Experiments comparable to those of Figs. 5d and 5e with components II, II’, and II” were performed (Figures 9B and D). The results in this analysis did not differ from those obtained with the standard procedure. The proteins show complete immunological identity.

Isoelectric Focusing A series of experiments was performed

to test if some of the observed isoelectric focusing bands were due to degradation or interaction of the subunits. Staining for copper (18) shows that all bands seem to bear the metal. Major bands observed for the whole stripped preparation correspond to those observed for the purified fractions (Fig. 4, column G). Focusing the purified subunits of Zone II (II, II’, II”, IIa, and IIa’) and Zone III (IIIa and IIIb) individually did not result in a decrease in the number of bands relative to the results shown in Fig. 4. Changes in the number of bands were not observed when the zones were analyzed individually or in mixtures. In a series of experiments the bands from a focused sample of whole stripped hemocyanin were cut from the gel, eluted, and rerun. The total number of bands observed on the refocused samples did not

IIldE)lllbIUb

IY Y Yb

FIG. 10. Relationship of row 1 initial chromatographic isolation; row 2, immunoelectrophoretic identification; row 3, alkaline-gel electrophoretic band identification, and row 4, SDS-electrophoresis identification. The brackets over IIa and IIIa, and V and (V’-VI) in row 2 indicate immunological cross- reactivity.

758 BRENOWITZETAL

differ from whole stripped hemocyanin (data not shown). No major differences in the isoelectric focusing pattern appear under different experimental conditions. The addition of 8 M urea to the gels results in some smearing of the bands, even after a 12-h focusing point, although the general pattern is the same as in the control gels. No reduction of the number of bands is observed. Prior to the run with urea, all samples were denatured directly in 8 M urea, 20 pM DTT or 6 M guanidine-HCl, 20 mM DTT, and then dialyzed vs the final urea solution. Addition of reductant, such as DTT, does not alter the major isoelectric focusing bands. To counteract possible ionic interaction, protein-protein or protein-ampholyte interactions, the ionic strength of the gels was increased by add- ing 0.5 M glycine. The additive did not modify the banding pattern of the proteins. As far as interaction with ampho- line is concerned, the same banding pattern appears when the samples are run in wide or narrow pH ranges, on prefocused or nonprefocused gels, and when samples are applied to the cathode or the anode.

DISCUSSION

Figure 10 presents a correlation of the chromatographic separation and the com- bined immunological and electrophoretic identification of the subunits. The differences in the identification of subunits by immunoelectrophoresis and polyacrylamide electrophoresis can be attributed to the higher resolution of charge isomers by polyacrylamide gels and isoelectric focusing and the additional structural infor- mation detected by immunoelectrophoresis. The extensive control experiments have led us to conclude that the electrophoretic heterogeneity observed is not ar- tifactual. The resolution of proteins by polyacrylamide electrophoresis is due to a combination of factors including net charge, conformation, and molecular weight. Comparison of previous studies (1,6) with those presented here shows that the apparent number of subunits increases with the resolution of the analytical sys- tem. Electrophoretic studies of human hemoglobin variants, where the location of the amino acid substitution in the three-

dimensional structure is known show that it is unlikely that all isoproteins would be detected with a single electrophoresis protocol (19). The limitations of using a single electrophoresis protocol suggest our data represent a lower limit estimate of the electrophoretic diversity. However, the similarity of the polyacrylamide electrophoresis and the higher-resolution isoelectric focusing patterns suggests that a further increase in the electrophoretic identification of subunits is not likely.

With the exception of subunits II, II’, and II”, there is a close correspondence between observing a single band on SDS- electrophoresis and a single precipitation peak by immunoelectrophoresis. Im- munoelectrophorically identical subunits, which are distinguished on alkaline electrophoresis, such as I, I’, I”, IIa, IIa’; IIIb’; and V, V’ do not show any heterogeneity on SDS-gels (Fig. 10). However, differences in apparent molecular weight are observed between immunologically unre- lated subunits such as I, II, IIa, and IIIa, etc. The two monomers of the dimer (VI- VI), which show partial immunological identity, each correspond to unique bands on SDS-electrophoresis (Fig. 3M). This is also observed for IIa, which is immunologically related, but deficient, to IIIa. IIa has a lower apparent molecular weight than IIIa which suggests that a portion of IIIa is “trimmed off,” possibly during post-translational modification, to pro- duce IIa (8) (Fig. 10).

The differences in apparent molecular weight between subunits on SDS-gels suggest differences in the chain length of the immunologically distinct polypeptide chains. However, it must be considered that carbohydrate, single amino acid substitutions, incomplete denaturation, and differing degrees of detergent binding all may influence the apparent SDS molecular weight (20-22).

The alkaline electrophoresis resolved a total of 15 bands, an increase over the 12 bands reported by Mark1 et al. (6). The difference in our results and Markl’s is probably due to the different pH’s (8.9 and 9.6) of the electrophoresis protocols used in the two studies. Of the 15 electropho- retie bands, 8 can be distinguished im-

SUBUNIT HETEROGENEITY OF Lamuhs HEMOCYANIN 759

munologically with two pairs of subunits, IIa and IIIa, and V and VI, showing cross- reactivity (8). The absence of immunological cross reactivity between subunits suggests that there are no determinants in common, and that the outer surfaces of non-cross-reacting subunits are different from each another. This implies that each class of subunits would have a distinct set of intersubunit interactions in the native molecule. The electrophoretic heterogeneity of immunologically identical components, such as in the set I, I’, I”, and the set II, II’, and II”, suggests the presence of charge isomers which have identical surface topography. If a charge substitution is not in a critical location, the electrophoretically distinct but immunologically identical subunits would be expected to have identical assembly properties.

An example of the latter phenomenon was found in a study of Limulus and Tuchvpleus subunits (8). Immunological cross-reactivity is found between the subunits of these two species of horseshoe crabs. However, a charge change on one subunit throws the subunit correlations out of electrophoretic order.

Evidence that electrophoretic differences are not always indicative of altered function is found in the study of human hemoglobin variants. There are variants where the (Y chains of HbA are different in charge but still form CQ-& tetramers with little or no functional difference (23). Examples of the other extreme, that of extensive modification of properties due to single amino acid substitutions, are hemoglobins Chesapeake and Kansas whose substitutions result in high- and low-affinity oxygen binding, respectively (24,25). The critical factor determining the effect of a substitution is the location of the substitution in the protein’s three dimensional structure.

Reassociation studies with partially purified subunit preparations of Limulus subunits have shown that the different subunits play different roles in stabilizing the native molecule (3). Similar experiments, with immunologically and electrophoretically pure subunits, confirm the varied roles of specific subunits in assembly and also show that immunologically

identical, yet electrophoretically different, proteins can substitute for each other in assembly. Correspondingly, distinctly different assembly characteristics are observed among the immunologically noni- dentical subunits (M. Brenowitz and C. and J. Bonaventura, in preparation). The same pattern of the immunological identity of a subunit mirroring its structural and functional properties is seen in the affinity and allosteric modulation of the oxygen-binding properties of the subunits.

Subunits II, II’, and II” are immunoelectrophoretically identical, yet appear distinct on alkaline gels, SDS-gels, and by isoelectric focusing. Moore and Riggs (16) reported identical two-dimensional tryptic peptide digest maps for II, II’, and II” (designated II(l), 11(2), and 11(3), Ref. (15)). Carbohydrate assays on the zone II proteins, as well as the other zones, are negative (Moore and Riggs, personal communication). The identical peptide maps and the immunological identity indicate that the differences in these proteins are minimal. The most likely possibility to account for the differences observed on both native alkaline and SDS-gels is a one or two amino acid substitution not detected on the digest maps, yet sufficient to alter the electrophoretic mobility of the proteins.

The concept that immunologically identical subunits have similar tryptic digest maps, and that immunologically different subunits have different patterns, has been found to hold for a number of other subunits. For example, subunits V and (V’- VI), which partially cross-react (8), have very similar digest maps, though they differ significantly in about five spots. This also holds true for IIa, which is cross-re- active, though immunologically deficient, with regard to IIIa. The digest maps of subunits with no immunological cross- reactivity are clearly distinct from one another (Moore, personal communication).

Despite the intense study given Limulus hemocyanin it still is uncertain why a 48- subunit aggregate is formed and what the specific relationship of subunit heterogeneity to oligomer formation is. Determi- nation of the X-ray diffraction structures of subunit II of Limulus and Panulirus


interuptus hemocyanins reveal trigonal antiprism symmetry for the hexamer (15, 26). Based on this symmetry for the hexamer and on electron micrograph profiles of the larger aggregates Klarman et al. (27) have predicted that there are five nonequivalent positions in the Limulus 48- subunit aggregate. That there are in fact more structurally distinct subunits is con- sistent with the hypothesis that there is a relationship between the degree of heterogeneity and the extent of aggregation.

However, comparison of these results with the two closely related species Eu- rypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, is not compatible with the proposed relationship between the degree of heterogeneity and the extent of aggregation state (6, 28, 29). Both of these proteins aggregate only to the 24-subunit level yet have the same degree of immunologically identified subunit heterogeneity with Limulus hemocyanin. This suggests that the argument for subunit complexity as a requirement for higher aggregation state does not hold true for the 24- to 48-subunit step, which is unique for horseshoe crab hemocyanin. The 24- to 48-subunit aggregation by Lim- ulus is a calcium mediated step that does not induce a change in the functional properties upon aggregation (30). Thus only minor subunit modifications may have been required to allow two 24-subunit aggregates to bind.

This study is a necessary first step in the systematic investigation of the subunit interactions in the Limulus hemocyanin oligomer. Although the properties of the oligomer are not simply the sum total of the properties of the subunits, the mechanisms for cooperative oxygen binding, the reverse Bohr effect, and the an- ionic regulation of the allosteric constants must all result from specific subunit interactions within the oligomer. Through this detailed study of the nature of the constituent subunits and the differences among them, we have provided a frame- work for further inquiry into the interactions and pathways by which they as- semble into the native oligomer.

ACKNOWLEDGMENTS

We thank Margaret Moore for communication of unpublished data and for a critical reading of the manuscript The initiation of this study was stimu- lated by discussions with Dr. Jurgen Markl. M.B. wishes to thank Drs Jean and Josette Lamy and Michele Leclerc for their hospitality and instruction in immunoelectrophoresis. This work was supported by NIH Grants HL 15460 and ES0 1908 and NSF Grant PCM 7906462

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Documents

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin