J. Mol. Biol.

(1984) 174, 319-341

Structure of Haptoglobin and the Haptoglobin-Hemoglobin Complex by Electron Microscopy

.JOHN C. WEJMAN~. DAVID HOVSEPIAN~,JOSEPH S. WALL',JAMES ANDJONATHAN GREER't F. HAINFELD

1Department of Biological Sciences,Columbia University New York, N. Y. 10027, U.S.A. e t o Biology, Brookhaven National Laboratory A 2Departm n Upton , N.Y . 11973, U.S .. (Received 20 July 1983)
The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 M,. In the complex, each H chain binds an afl dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124( + 8) A and the interhead distance is 87( + 7) A. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal. Thus, the a/I dimers are binding to the H chains, but off the long axis of the barbell by 127 in a tran.sconfiguration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5( + 6.3) a or 30 A greater than for haptoglobin. The N terminus of the /? chain was located on the tram off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the afi dimers in the region of the fi N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the afI dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the a/?dimer to the H chain of haptoglobin.

1. Introduction
Haptoglobin (Putnam,
t Author Laboratory. 0022

Javid, 1978). Haptoglobin has the remarkable property that it forms a stable, effectively irreversible complex with hemoglobin
to whom all correspondence should be addressed. Present address: Physical tlbbott Laboratories. Abbott Park, Nort,h Chicago, IL 60064, U.S.A. 3 19 2836/84/100311)-23 $03.00/O Biochemistry

is a serum 1975; Pintera,

protein 1971;

that

is present

in all mammals

and vertebrates

0 1984 Academic Press Inc. (London) Ltd.

320

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that has been released from erythrocytes in the circulation. This complex is removed from the blood stream in the liver, digested there, and the iron recycled. Based upon this very strong binding, it has been suggested that Hpt is involved in recycling of Hb iron, although Eaton et al. (1982) have proposed that it may function as a bacteriostat to deny heme iron to pathogenic bacteria. Hp is composed of two types of chains, a light (L) chain and a heavy (H) chain, which are now known to be synthesized as one polypeptide and then processed to form the two chains (Haugen et al., 1981). All species of animals that contain Hp have a H chain of -245 residues plus carbohydrate with a molecular weight of N 33,000 (Kurosky et al., 1980). Virtually all these species have an w 83-residue L chain (Kurosky et al., 1980; Black & Dixon, 1968). The H and L chains are connected by a disulfide bridge. In addition, the HL unit is joined to another HL via a disulfide bridge between the L chains to form the complete molecule of -85,OOOM,, which can be symbolized as H-L-L-H (Black & Dixon, 1968). In humans, this form of Hp is called Hpl-1. The interaction of H chains with L chains and the assembly of Hpl-1 from the isolated chains have been studied extensively (Bernini & Borri-Voltattorni, 1970; Valette et al., 1981: Malchy & Dixon, 1973). Hb is also a tetramer, composed of two a and two /I chains. An atomic model of the Hb structure has been available for some time from X-ray crystallographic studies (Baldwin, 1980; Ladner et al., 1977). From this structure, Perutz showed that there were two types of interactions between a and /I chains, which he called aJ1 and a& (Perutz, 1965). When the Hb tetramer splits into two dimers, the alp2 interface is exposed and the alpI interface is preserved. These dimers are often referred to as a& dimers. A variety of studies have shown that Hb interacts with Hpl-1 as two al/l1 dimers to form the Hp-Hb complex (Cxl-1) (Laurel], 1959; Nagel & Gibson, 1967,1971; Peacock et al., 1970; Hwang & Greer, 1979,198O). Each aJll dimer binds to a H chain of Hp; the L chain does not interact with Hb at all (Valette et al., 1981; Bernini & Borri-Voltattorni, 1970). From the fact that ab dimers but not tetramers will bind to Hp, and from the observation that many different species of Hb will bind to human Hp, it has been suggested that the sequence-invariant a& interdimer interface is probably the region of Hb that binds to the H chain (Nagel & Gibson, 1971). Sequence analysis of Hp (Kurosky et al., 1974,1975,1976,1980) has shown that the H chain is highly homologous to the mammalian serine proteases. This strong homology has permitted the construction of a three-dimensional model of this protein by comparative model building methods (Greer, 1980,1981). Biochemical studies have determined that the vestigial protease active site area of the H chain does not bind the a/? dimer (Arcoleo & Greer, 1982). Modification studies using selective proteolysis have demonstrated that the Hb binding region on H chain lies around residues 130 to 1371: which corresponds to the region about the autolysis loop in the homologous chymotrypsin structure (Lustbader et al., 1982).
7 Abbreviations used: Hp, haptoglobin; Hb. hemoglobin; Cx. haptoglobin-hemoglobin STEM, scanning transmission electron mivroscope(y). 1 The residue numbering used here is for the H chain. The detailed correspondence chymotrypsinogen sequence, which is commonly used in comparing the various homologous protease sequences, is given by Qreer (1980,1981). complex; to the swine

STRUCTURE

OF HAPTOGLOBIN-HEMOGLOBIN

32 I

Thus, much is known about the structures of Hb and Hp as well as about the regions on these two molecules that interact in the complex. However, no structural information is available about the relative orientation of these two molecules in the complex or about the detailed structure of the very stable combining site. Furthermore, nothing is known about the three-dimensional structure of the L chain or about its interaction with the H chain and with anot)her L chain to form the Hpl-1 molecule. We began to examine Hp in the high-resolution scanning transmission electron microscope in order to investigate the extent to which the shape and structure of a molecule as small as Hpl-1 and Cxl-1 could be determined using electSron microscopy. In this paper, the results of these studies are reported. We present new information about the structure of Hpl-1, its component chains, and its interaction with Hb. We show that a landmark site on the complex molecule can be localized using suitable labeling experiments. The identification of several such sites on the Hb afi dimer and on the H chain should eventually allow the determination of the relative orientation of these two molecules in the complex and a detailed description of the interaction site.

2. Materials
(a) Preparation

and Methods
Hb and Cxl-1

of Hpl-1,

Human Hpl-1 was isolated according to the procedures of Waks & Alfsen (1966). Further purification was achieved by repeated chromatography on columns of Ultrogel AcA 44 (LKB) until all traces of contaminant protein, mainly albumin at lower molecular weight and ceruloplasminat higher molecularweight, wereremoved. The molar extinction coefficient used for Hpl-1 was E = 10.2 x lo4 at 280 nm. Hb was isolated according to the method of Yip et al. (1972). For Hb in the cyanomet form, the extinction values of 11 x lo3 at 540 nm, 12 x lo4 at 420 nm. and 2.9 x lo4 at 280 nm per heme were used. The complex between Hpl-1 and Hb was prepared by mixing 1.5 mol Hb per mol Hpl-1. This mixture was then run on an AcA 44 molecular sieve column to separate excess unreacted Hb from the Cxl-1. The concentration of Cxl-1 was measured by quantitating the Hb in the complex.
(b) Preparation The cross-linked human hemoglobin of Cxl-1 using cross-linked (HbXL) was generously Hb provided by Drs Reinhold

and Ruth Benesch, and was prepared by them as described(Beneschet al., 1975). The
cross-linker was 2-nor-2-formylpyridoxal 5-phosphate (NFPLP), where the 2 and 4 carbons of rJFPLP are joined to the amino nitrogens of Lys82fl, and Vallb,, respectively (Arnone et al., 1977). As a result, the 2 afi dimers of the Hb tetramer have been connected covalently. Since both HbXL and Hpl-1 have 2 combining sites for each other, a variety of crosslinked Cx 1- 1 polymers of the form: H-L-L-Ha/$(-BaH-L-L-Ha/?-),-BcrH-L-L-H, can be prepared as described stoichiometries were adjusted to equivalents of Hpl-1 were mixed under CO. One molar equivalent unoccupied H rhain Hb-binding n. = 0. 1, 2. 3, the relative Two molar temperature to cap the the solution by Benesch et al. (1976). In this case, favor the formation of dimer (n = 0, above). per mol HbXL, incubated for 24 h at room of non-cross-linked Hb was then added sites at the ends of each polymer chain, and

321 was incubated an additional of Cxl-1 and the series:

J. C. WEJMAN 30 min. The solution

ET AL. contained a product mixture consisting

abH-L-L-Ha/l-($aH-L-L-H@-),-BaH-L-L-H/la,

?z = 0, 1, 2, 3,

The sample was then passed through an AcA 34 sizing column. The non-cross-linked Cxl-1 was completely separated from the polymers on this column and discarded. In addition, the various polymers could be resolved into a dimer-enriched portion and a mixture of higher polymeric forms, as determined by polyacrylamide gel electrophoresis under native conditions using a 5% Laemmli gel system (Laemmli, 1970). (c) Preparation of specimens and electron microscopy

The procedures followed were essentially as described in detail by Mosesson et al. (1981). Protein solutions were applied to the carbon surface of the grid by a minor modification of the flotation technique (Estis & Haschemeyer, 1980). Standard solutions of Hpl-1, Cxl- 1, and crosslinked Cxl-1 were diluted to a final concentration of 1 to 5 pg/ml immediately before application onto the carbon film grids. Attachment times varied from 30 s to 2 min. Grids were usually washed twice with distilled water at 30 s intervals and then washed a third time with water containing tobacco mosaic virus (provided by P. Day) that had been freshly chromatographed on a Sephadex G-100 column in distilled water. Negative staining was accomplished by including a final wash with 2% (w / v ) uranyl sulfate (Polysciences, Inc., Warrington, Pa.) that had been prepared freshly and centrifuged at 100,000g for at least 5 min before use (-45 s drying time). Freeze-drying was carried out as described by Mosesson et al. (1981). Specimens were imaged at the Brookhaven STEM Biotechnology Resource (Wall, 1979; Mosesson et al., 1981). The STEM samples were mounted in the microscope with the molecules above the substrate. However, the images are presented in the micrographs as if they were viewed from below, i.e. through the substrate. The micrographs were transferred digitally to magnetic tape for analysis off-line and were photographed from a highresolution cathode ray tube display using Polaroid 665 positive/negative film. The magnification used in most cases was 251,000 x with occasional micrographs taken at onehalf and twice this value. The electron flux at this magnification was around 50 e/A. (d) Dimensional analysis of STEM micrographs

Micrograph negatives were projected on the screen of a Nikon Shadowgraph (model 6C) giving a IO-fold magnification. The center of mass of a particular density feature on the micrograph was estimated by eye. Distances were measured by positioning the initial point at the screen center, which was defined by the intersection of cross-hairs, then translating the destination point to the center using X and Y co-ordinate knobs, which moved the image platform in gradations of 0.02 mm. The measured distances were converted to BngstrGms using the appropriate scaling factor for the magnification, usually 40.25 A/mm.

3. Results
(a) The structure Hpl-1 is quite difficult to visualize of Hpl-1 electron microscope for several

in the

reasons. The major impediment is that Hpl-1 adheres very poorly to the electron microscope grids. Consequently, after repeated attempts, only a relatively small number of molecules have been observed. In addition, since the molecular weight of Hpl-1 is 85,000, it is much smaller that the protein aggregates or molecules that are usually examined by electron microscopy. Therefore, in order to observe

STRUCTURE

OF

HAPTOGLOBIN-HEMOGLOBIN

323

FIG. 1. STEM micrographs of negatively stained specimens of Hpl-1 and &l-l. The micrographs were originally taken at 251,ooO x and are shown here at 550,000 x Row 1: Hpl-1 molecules at the center in each field. Rows 2 to 4: (31-1 molecules at the center in each field. Row 2: last field shows 2 molecules aligned. In some of these molecules, the bilobal nature of the ellipsoidal head groups is more evident.

t,he structure of this molecule in any detail, the negative staining had to be optimal. Figure 1 (row 1) presents several examples of Hpl-1 The molecule is composed of two approximately spherical head groups connected by a relatively thin filament, usually with a small knob in the very center. Approximate dimensions of these features are given in Figure 2(a). The spherical head groups have a diameter of 37( f5) A (measuring 8 molecules). The overall length of the molecules is 124( +8) A (8 molecules). The small knob and filament in the center were harder to measure accurately in these molecules, but were around 25 and 15 A in diameter, respectively. The overall shape of the molecule is thus quite elongated and resembles a barbell.

J.

C. WEJMAN

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FIG. 2. Schematic representation of (a) Hpl-1 and (b) Cxl-1, derived from the micrographs. Unspecified L chain dimensions in Hpl-1 were drawn using the dimensions from Cxl-1 specimens and are in agreement with the approximate dimensions (< 10 molecules) obtained from the sparser Hpl-1 micrographs. The points labeled 1 through 4 are those measured for the purpose of calculating the off-axis angle of the aj dimer (see the text).

(b) Hpl-1

complexed

with Hb

The structure of Cxl-1 was examined next. In contrast to Hpl-1, Cxl-1 adhered readily to the electron microscope grids. Several examples of these molecules are presented in Figure 1 (rows 2 to 4). Once again, a two-headed structure was observed, which had an interconnecting thin filament with a central knob. The central knob was measured to be 25( f5) w in diameter (30 molecules) and the connecting filament was about 14( & 1.5) a (5 molecules) in diameter. The major difference observed in Cxl-I relative to Hpl-1 was that the formerly spherical head groups at the two ends of the molecular barbell were now significantly larger and clearly ellipsoidal in shape. When the molecule was ideally stained, the head groups appeared to be composed of two attached lobes (see especially Fig. 8, where the molecules on the right show head groups in which the bilobal effect is very prominent). The additional density that extends beyond the original spherical head group observed in Hpl-1 must correspond to the c$ dimer. A schematic view of the Cxl-1 molecule together with approximate dimensions appears in Figure 2(b). In order to analyze the differences between Hpl-1 and Cxl-1 quantitatively, the interhead group distances were measured on a large number of molecules and a histogram was plotted for the two forms (Fig. 3(a) and (b)). The mean Hpl-1 interhead group distance, measured from center to center, is 86+3(f 6.9) A (17 molecules), while that for Cxl-1, center to center of the ellipsoids, is 116.5( f 6.3) ,& (100 molecules). Fewer molecules were available for Hpl-1 than for Cxl-1 because of the greater difficulty in obtaining good images of the Hpl-1 species, as described above. Nevertheless, the difference of -30 I( is clearly reproducible.

STRUCTURE

OF

HAPTOGLOBINgroup 100 I I distance

HEMOGLOBIX (8) I20 I 14

1

Interhead 0.4 (a) 80 I

0.2

0 (b)

g 2

0.2

0.2

I 2.0
Interhead

I
2.5 group distance

I
3.0 (mm) .

FIU. 3. Histograms of the interhead group separations for (a) Hpl-1, (b) negatively stained Cxl-1 (c) freeze-dried &l-l. Arrows indicate the mean values. The abscissa below shows lengths in mm (for 251,000 x magnification) as measured on the original micrograph negatives. These values are converted to Bngstrb;m units on the upper abscissa scale. The ordinate (IV/~V,) shows the number of specimens (A) at each length as a fraction of the total population (-V,). The values of X0 are given in the text.

A close examination of many of the images of Cxl-1 showed that the additional densities for the Hb a/3 dimers of both head groups were lying at an angle to the molecular barbell axis (Fig. 1, rows 2 to 4). In most of the molecules, the t$ dimer density extended on opposite sides of the molecular axis in a trans configuration. Tn order to evaluate this observation more systematically, all molecules in the Cx l-1 micrographs that were clearly isolated from their neighbors and in which the stain permitted clear definition and delimitation of the head groups ( N 50% of the population) were examined. Of these 157 molecules, 80% were classified as trans. 12% as cis, and 8% as linear. To further describe the off-axis binding of aP, the angle that the c$ dimer makes with the molecular axis was measured for the more clearly defined molecules as follows. The density for a typical bilobal head group was fitted to each ellipsoidal head group on a molecular image. The positions of the center of the UP lobe, of the inner lobe, and of the two junctions between the thin filament

3%

J. 0.4 IO)

C. WEJMAN

ET

AL

100

120
Off-axis

140
angle, &

160

I IO

FIG. 4. Histograms of the off-axis angle, cp,,, between the ellipsoidal head group and the H-H axis of Cxl-1 (see Results, section (b) for a description of the measuring technique). (a) Histogram for native Cxl-1; (b) histogram of the off-axis angle for the Cxl-1 molecules in the crosslinked dimer and polymer samples. The abscissa is expressed in degrees.

and the two head groups were measured in the molecule (these positions are labeled 1 through 4 in Fig. 2(b)). The off-axis angle, q,, was then taken to be that which is formed by the two vectors (X,-X,) and (X,-X,). A histogram of the values obtained for this angle appears in Figure 4(a). The average value of the offaxis angle was 127.4 + 11 (40 measurements). One other interesting observation is worth mentioning here. Since most of the Cxl-1 molecules have the u/I dimers binding off the main axis in a trans configuration, there are two possible ways in which these molecules can lie on the grid: either like an S or like a Z (see Fig. 1, rows 2 to 4). When the 128 molecules in the trans configuration were examined, 118 were found to be in a Z configuration and only ten were S-like, giving a ratio of 12 : 1 of Z over S. (c) Studies of freeze-dried preparations of Cxl-1

1-n order to check on the reliability of negatively stained images of Hpl-1 and Cxl-1, freeze-dried specimens of Cxl-1 were also examined (Fig. 5). The molecules exhibited the same basic morphology as the negatively stained specimens, but less detail was apparent. Two head groups are observed, but the connecting filament and central knob are only occasionally visible. The ellipsoidal or bilobal shape of the head groups is not readily discernible nor is the trans off-axis configuration of the a/3 dimers. A similar decrease in detail in freeze-dried samples relative to negatively stained specimens has been observed in a number of other systems (Cohen et al., 1979; Woodcock et al., 1980; Mosesson et al., 1981). It is likely due to the reduced contrast of the freeze-dried preparations and their greater sensitivity to radiation damage during exposure to the electron beam. The interhead group distance was measured for the freeze-dried Cxl-1 preparation (Fig. 3(c)). The mean value of the interhead group distance was 107.1( & 18.3) A (42 molecules). This value is - 8 A shorter than that measured for

STRUCTURE

OF

HAPTOGLOBIN-HEMOGLOBIN

327

FIG. 5. Micrograph of freeze-dried Occasionally, the connecting filament 251,000 x ; here it is 630,000 x

Cxl-1. The pairs of head and central knob are discernible.

groups are The original

clearly apparent. magnification was

negatively stained Cxl-1. The probability that the two sets of measurements are different samples of the same underlying distance distribution is less than 0.001 by the t-test (Sokal & Rohlf, 1973); therefore, the difference between the freezedried and negatively stained interhead group mean distances is small but significant. This may be due to small systematic differences in the determination of the center of mass, preferential negative staining of certain areas of the molecule, or differences in conformation under high and low salt conditions. (d) Cross-linking studies on Cxl-1
a

The reaction of HbXL with excess Hp (see Materials and Methods) generated series of polymers of the form H-L-L-Haj?(/?aH-L-L-Ha/Q&H-L-L-H,

328

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C. WEJMAN

ET AL.

23

10

FIG. 6. Laemmli 5% polyacrylamide gels of crosslinked Cxl-1. The gel was stained with hemesensitive benzidine peroxide, and so reveals only the bands that contain hemoglobin. Lane 1, HbXL with a 2-fold molar excess of Hpl-1; lane 2, like lane 1, but with a subsequent addition of excess native (non-crosslinked) Hb. Lanes 3 to 10, sequential fractions from an AcA34 sizing column elution of the sample shown in lane 2. Lane 3, fraction enriched for crosslinked Cxl-1 polymers; lane 4, fraction enriched for crosslinked Cxl-1 dimers, the major band in this lane is the Cxl-1 dimer; lanes 5 to 8, fractions containing non-crosslinked Cxl-1; lanes 9 and 10, fractions containing the excess, noncrosslinked, capping Hb.

n=0,1,2,... (Fig. 6, lane 1). The molecules were capped with native Hb (Fig. 6, lane 2), then run on an AcA34 sizing column to produce fractions enriched for crosslinked dimers (Fig. 6, lane 4) and higher polymeric forms (Fig. 6, lane 3). Figure 7 shows a number of the images obtained for the crosslinked Cxl-1 dimer-enriched fraction. Each image is composed of two typical barbell-shaped Cxl-1 molecules, with two of the ellipsoidal head groups, one from each barbell, in close apposition, marking the site of the crosslink. Higher polymeric forms of crosslinked Cxl-1 are shown in Figure 8. These are chains of connected Cxl-1 barbells, attached end to end. AS a check to be sure that the Cxl-1 molecules in this crosslinked sample were normal, the interhead group distance was measured. The mean value was 115.0( k9.1) A (34 molecules). While the distribution is a little broader, the mean value is virtually the same as that of native Cxl-1. In order to determine the position of the crosslinker on the Cxl-1 molecule, several measurements were made. For each crosslinked pair of molecules, the coordinates of the center of mass of the two apposed, crosslinked head groups were measured and the distance between them calculated. The mean crosslinked head group center to center distance was 65*9(f 10.9) A (18 molecules). For those molecular pairs where the two apposed head groups were clearly ellipsoidal or bilobal, a typical bilobal head was fitted to each head group and the positions of the two crosslinked ajl dimers were measured. This allowed the halculation of the distance between the apparent centers of the two crosslinked a/? dimers. The mean inter-a/I chain distance was 58.0( + 7.8) A (13 measurements). Next, the angle, cpc,that is formed between the two connected head groups and

STRlJCTlJRE

OF

HAPTOGLOBIN-HEMOGLOBIN

3!!

FIG. 7. Micrographs of negatively stained specimens of crosslinked Cxl-1 the predominating inside to inside configuration; row 4, left illustrates a row 4, right presents a roll-over configuration, which is inside to outside; row to outside configuration, it is actually formed from 2 &-shaped molecules. defined in the text and are shown in Fig. 11. Magnification is as in Fig. 5.

dimers: rows 1 to 3 show cross-over configuration: 5, left is the only outside These configurations arc

each of the distal head groups (Fig. 9(b)) was measured for each of 18 crosslinked pairs of Cxl-1. Two such angles occur in each pair of crosslinked molecules. The results are presented in Figure 10. The two crosslink angles for each connected pair of Cxl-1 molecules are plotted as a function of the absolute value of the difference between the two measured angles (Fig. IO(a)). The mean crosslink angle was determined to be 97+32 (36 measurements). An explanation for the wide breadth of this angle distribution appears in the Discussion, section (c). The crosslinked molecules were then examined to see if the density of the head group ellipsoids that corresponded to the Hb a/3 dimer appeared off-axis, as in

J.

C. WEJMAN

ET AL.

FIG. 8. Micrograph of crosslinked Cxl-1 dimer (right) and polymer (left). A schematic interpretation is shown below. The bilobal nature of the head groups is clearly evident in the dimer molecules. Note that the dimer is clearly inside to outside (see the text and Fig. 11). The polymer was interpreted by using the standard &l-l interhead distance and the characteristic proximity of crosslinked pairs of head groups. The especially intense head group represents the overlap of two crosslinked head groups from adjacent molecules, The original magnification was 251,000 x ; the molecules are shown here at

2900$00x.

native Cxl-1. The off-axis angle, cpO,was measured for these molecules as described above. The resulting histogram appears in Figure 4(b), where it is compared to that obtained for native Cxl-1. The mean value obtained for the crosslinked Cxl-1 was 138 f 2 1.5 (44 measurements), which is higher than the 127*4& ll*O found for Cxl-1. However, the histogram for the crosslinked molecules shows that the angles for most of the molecules fall in the same range as those of native Cxl-1, while only six of the 22 crosslinked molecules gave all the unusually high angles. When these six molecules are removed, the mean value of the angle and the distribution of angle values is indistinguishable from that found for native Cxl-1. These six molecules were examined in the electron micrographs, and in each case they were found to be especially prone to distortion. For example, in one case both members of the crosslinked pair were in the cis configuration, which seemsmost likely to be a result of distortion, since virtually all the rest of the molecules are trans. In another case, the distorted molecule lies

STRUCTURE

OF

HAPTOGLOBIN-HEMOGLOBIN

33 I

Fm. 9. Schematic drawings defining the polar co-ordinate systems used for Hpl-1 and (x1-1. (a) These polar co-ordinates are used to describe the direction of the binding of the a/l dimer (dotted circle) to the H chain (continuous circle). The off-axis angle of the a/? dimer, cp,,, is defined as shown. The z-axis (q, = 0) is aligned along the line connecting the centers of the head groups. The p and pO values limit the possible direction of the a/m-H chain contact to the broken circle shown. One possible direction for the as dimer is represented by the arrow pointing to the dotted circle. (b) The polar coordinate system used to define the location of the /I N terminus in the Cxl-1 molecule. The crosslink angle, cp., is shown for both molecules of this crosslinked pair. The z-axes are aligned to the centers of the ellipsoidal/bilobal head groups. The z-axis is defined for one of the Cxl-1 molecules and points in the direction in which the aB dimer extends off the molecular barbell axis.

(a)

1
*

120
I l

2 a -

60.
. * . -

40.
=-

0-U

b)

Iii
40

II5
160

Pm. 10. Distribution of the crosslink angle, qe. (a) The 2 crosslink angles (filled circles) for each Cxl-1 dimer are shown connected by a straight line. The absolute difference between these angles is plotted along the ordinate. This difference represents the degree to which the axes of the 2 crosslinked (xl-1 molecules are not parallel (see the text). (b) Histogram of the crosslink angle. The mean value is 96.7. The standard deviation of 31.9 is very broad and is explained in the Discussion, section (c).

332

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C. WEJMAN

ET AL.

(0)

(b)

(cl

representations of the 3 possible crosslinked Cxl-1 configurations, assuming that q, = 96.7 and that the Cxl-1 molecule lies in a Z or S configuration. (a) Inside to inside; (b) inside to outside; (c) outside to outside.

FIG. 11. Schematic

in the middle of a higher molecular weight crosslinked polymer and probably has been rotated as a result of the constraints imposed by these neighboring molecules, so that both off-axis angles of this molecule are unusually high. Thus, the crosslinked Cxl-1 molecules display a similar off-axis angle distribution to native Cxl-1, even though they have been selected for their crosslinking and not for their off-axis angle. The preference for the Z over the S orientation occurs once again in the crosslinked Cxl-1 molecules; however, the ratio, 27 to 13, is lessmarked than that of native Cxl-1 . When two trans configured Cxl-1 molecules are crosslinked via the Hb dimers, with a mean crosslink angle of 97, there are three possible ways in which Z or S-shaped molecules can interact: so that the connection is between the inside curve of the Z of one molecule with the inside curve of the other molecule, inside to outside, and outside to outside (seeFig. 11). In all, 20 crosslinked Cxl-1 dimers were examined. Of these, 16 were inside to inside, three were inside to outside, and only one was outside to outside.

4. Discussion
(a) The structure of Hpl-1 The electron micrographs of Hpl-1 show that the molecule consists of two spherical head groups connected by a filament with a central knob. Consideration of the massof the Hp subunits, 34,000 nlf, for the H chain versus 9000 M, for the L chain (Kurosky et al., 1980), and the linear arrangement of the interchain disulfide bonds between the subunits, H-L-L-H (Black & Dixon, 1968), indicates that each head group must be primarily the H chain, while the filament and central knob must be the two L chains. Further evidence supporting this assignment comes from the complex that is formed with Hb. Biochemical studies have shown that the a/I dimer binds exclusively to the H chain and that the L chain is not involved in complex formation (Valet&e et al., 1981; Bernini & Borri-Voltattorni, 1970). In the electron micrographs, the densities for the a/3 dimers, which have a molecular weight of

STKUCTUHE

OF HAPTOGLOBIB-HEMOGLOBIS

:%I

32,000, are clearly discernible and are attached at the outer edge of each head group. Thus, the head groups must correspond to the H chains. The structure of Hpl-1 that is observed is an elongated molecule with an axial ratio of 3.25 : 1 (Fig. 2(a)). This fits very well with the solution studies of Waks a,nd (<o-workers (Waks et al., 1969), who measured an axial ratio of 3 to 4 : 1 using small-angle X-ray scattering. However, rather than having an overall ellipsoidal shape, the electron micrographs indicate that the structure more closely resembles a barbell. Malchy et nE. (1973) have shown that the inter-L disulfide bridge can be reduced selectively in the intact Hpl-1 molecule. Experiments in our laboratory indicate that,, despite this reduction, the L chains remain strongly associated through non-covalent) interactions and will not exchange (J. C. Wejman & ,J. Greer. unpublished results). The presence of a knob of density of diameter about 25 a in the center of t.he Hpl-1 molecule is consistent with t.his observation and implies that the two L chains are interacting extensively at this point. H chain is homologous to the serine proteases and the known crystallographic structures of these molecules show them to be spherical with a diameter around 15 A. The overall shape of the H chain in the micrographs appears to be approximately a sphere as expected, but with a diameter of only 37 a. If anything, because of the additional carbohydrate in H chain, one would expect, the diameter to be even larger than in the known protease structures. The smaller value obtained here is presumably due to the fact that the micrographs are presenting a negative stain image of the molecule. This image may be considerably smaller than the true size because of the ability of the stain to penetrate the outer layer of the molecule. The micrographs give some glimpse into the structure of the L chain, about which little is known. The two connected L chains appear as a filament with a caentral knob. A single L chain is a cylinder with dimensions about 15 A by 25 ,A (Fig. 2). Stain penetration would tend to decrease the value of the diameter but not of the length of the cylinder, making the structure more isometric. Considering these dimensions and the small molecular weight of the L chain. -9000, this subunit is probably globular, as well. (b) Binding of Hb a/3 dimers to Hpl-1

Hb binds to the H chain as an aIfll dimer (Bagel & Gibson, 1971; Hwang & Greer, 1980). The position of the a/? dimer is seen clearly in the Cxl-I molecules, where the spherical head groups become ellipsoidal. In many cases, these elongated head groups appear to be bilobal, demonstrating the close H-a/I interaction. The addition of two Hb dimers at opposite ends of the molecule incrc>ases the mean interhead group distance from 86.8 A to 116.5 A, or by about 30 8. This value is consistent with the addition of a Hb afi dimer of mean diameter -30 a at both ends of the molecule, as shown schematically in Figure 2(b). The Hb afl dimer is an ellipsoid with approximate diameters of 30, 40 and 60 A. The shortest axis lies along the pseudo &-fold axis that relates the a chain to the fi

334

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chain in the dimer and is perpendicular to the a& interface. The longest axis runs from the tip of the a chain near the a-heme propionates to the tip of the fi chain near the P-heme propionates. The middle, 40 A axis runs between the a and /? chains in the aJll interface. Both the 40 A and the 60 A axes are parallel to the a& interface. The 30 A increase in interhead group distance in Cxl-1 over Hpl-1 suggests that the shortest dimension of the ~fi dimer lies along the interhead group direction. This orientation of the afi dimer would place the interdimeric alPz interface in position to interact with the H chain, as expected from biochemical experiments and from the invariance of the residues in this contact (Nagel & Gibson, 1971). The micrographs further show that the a/3 dimer is binding off the H chainH chain molecular barbell axis by cpO 127 (Figs 1 (rows 2 to 4), 2(b) and 4(a)). = A similar angle distribution was found for crosslinked Cxl-1 (Fig. 4(b)). This offaxis binding was deduced by Makinen et al. (1972), based on the electro-optical properties of the Hp-Hb complex. However, Makinen and co-workers were unable to distinguish by their method between a cis or trans relationship for the Hb dimers relative to the molecular axis. The electron micrographs show very clearly that the dimers bind to the substrate in a tram configuration relative to the H-H axis. It is important to realize that the distributions of the interhead group distances and of the off-axis head group angles are quite tight, with standard deviations of f 6.3 A and + 1l, respectively. Furthermore, the off-axis angular binding of Hb dimers is almost always trans to the molecular axis. These results imply that the attachments between the H chain and the Hb a/l dimer, the H and L chains, and the L and L chains must be quite rigid and that the Cxl-1 molecule is rather inflexible. The small number of caseswhere a cis arrangement is observed is likely to be due to distortion during binding of the sample to the grid. The few linear casesmay either be due to distortion or, more likely, represent a small fraction of molecules that are binding to the grid rotated about the molecular axis by &SO, an apparently less-favored position. The barbell shapes of Hpl-1 and Cxl-1 and, in particular, the tram off-axis binding of the Hb a/?dimers, imply that the Hpl-1 molecule has a 2-fold axis of symmetry relating its two L chains and two H chains. This axis would pass through the central knob of the molecule and its direction would lie perpendicular to the plane of the schematic molecule shown in Figure 2(b). The inter-L chain disulfide bridge connects the cysteine residues at position 15 in the two L chains. Since this bridge is formed between the same residue in the two L chains, it must lie on the molecular 2-fold axis and thus must be in the central knob of the molecule. It is striking that of the two possible ways that a trans configured Cxl-I molecule can rest on the microscope grids, the Z form appears overwhelmingly; that is, 12 to 1. When this result is compared to the very poor binding observed for Hpl-1 , it suggeststhat it is through the Hb a/? dimers that Cxl-1 is adhering to the grids. The preponderance of the Z versus the S form indicates that some particular region on the surface of the a/? dimer in the Cxl-1 molecule adheres preferentially to the grid. When the Cxl-1 molecule lies with its S-fold axis perpendicular to the microscope grid, i.e. in the Z form as is usually observed,

STRUCTURE

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HAPTOGLOBIN-HEMOGLOBIN

3%

,-J 184

Fm. 12. Stereo representation of the a-carbon plot of the model of H chain (Greer, 1980,1981), constructed from the serine proteases using comparative modelling methods. CyslOBH, which links the H chain to the L chain lies in the center at the very back of the molecule. Surface residues that fall on the cpO = 127 circle, as described in the Discussion, section (b), appear around the outer perimeter of the molecule

then both c$? dimers in the molecule have the same sticky surface contact with the grid. This double binding further explains why the Cxl-1 molecules are found primarily in the Z form?. The off-axis binding angle of the a/l dimer, cpO, can provide quantitative information about the site of Hb binding on the H chain. Since the H chain appears as an approximately spherical head group, it is convenient to define positions on its surface using a spherical co-ordinate system: p, 9, cp (Fig. 9(a)). In this case, p is the distance from the center of the spherical head group to the surface of the subunit; that is, its length is the radius of the H chain. The 40 angle is the off-axis angle, cpO, measured here to be 127*4+ 11.0 with the z-axis (cp, = 0) defined along the direction from the center of mass of the H chain of this head group to the equivalent point in the other head group of the molecule. Together, p and cp define a circle on the surface of the H chain, upon which the Hb binding site must lie (Fig. 9(a)). In order to specify a particular site on this circle, a 9 angle must be measured relative to an x-axis direction. 9 cannot be measured in this case, because there is no obvious feature of the H chain in the micrographs that can be used to distinguish an x-axis direction. The values obtained for p and cpOcan be applied to the model st,ructure for the H chain that was constructed based on its strong sequence homology to the mammalian serine proteases (Greer, 1980,1981; Kurosky et al., 1980: and Fig. 12). The I, chain binds to the H chain via a disulfide bridge to residue 105H in an homologous fashion to the disulfide bridge between the A and B chains of
t Note that the convention at the Brookhaven STEM facility produces the images as if viewed from below, i.e. through the grid. This fact must be considered when analyzing the structural implications of a Z versus S configuration and. in particular, when modeling the attachment site of the Cxl-1 molecule to the microscope grid.

33fi

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ET AL.

chymotrypsin (Birktoft & Blow, 1972). Residue 105H appears at the far end of the molecule as it is presented in this Figure, and the pseudo active site of the H chain lies at the front of the molecule in the center. If we assume that this disulfide bridge lies approximately on the H chain-H chain molecular axis, then a line from the CyslOSH to the molecular center of mass of the H chain defines the z-axis direction and the Hb dimer should bind at a cpO 127 angle to this line. = Kate that if the pseudo active site were involved in the binding, the cpO angle would be close to 180. Biochemical experiments have indicated that it is not involved (Areoleo & Greer, 1982) and the microscopy presented herein confirms this conclusion. A number of the surface residues of the H chain that lie at cpO 127 are labeled in Figure 12. Of these residues, those around 23H, 46H, 50H = and 80H can be excluded because carbohydrate chains are attached to the H chain at these sites, and they are known not to be involved in Hb binding (Rafelson et al., 1961). Studies in our laboratory using selective proteolysis (Lustbader et al., 1983) have found that residues in the region of 130 to 137H are implicated in the contact. This result fits very well with the 127 angle determined from the micrographs. (c) Location of th,e/I chain N terminus in the Cxl-1 structure The electron microscopic studies described above have established the general morphology of the Hpl-1 and Cxl-1 molecules and an approximate direction for Hb binding on the H chain. Electron microscopy can also be used to identify particular sites on these molecules. In this study, a chemically crosslinked Hb tetramer sample was used to make Cxl-1 that was specifically crosslinked between the a-aminogroup of Vall of one ,l?chain and the s-amino group of Lys82 of the second /3 chain (Arnone et al., 1977). $ Lmce the s-amino of Lys82 lies within 5 A of the N terminus of its own p chain, this crosslink can be considered to attach the two /I N termini to each other within the resolution limits of electron microscopy. The position of the crosslink, and hence the /? N terminus is indicated by the site of closest apposition of the ellipsoidal head groups of two crosslinked Cxl-1 molecules (Figs 7 and 8). A quantitative description of the position of the crosslink site on the ellipsoidal H/a/? head group can also be expressed in the spherical co-ordinates p, 3 and cp (seeFig. 9(b)). Here, p is the distance of the crosslink, i.e. the fl N terminus, from the center of the head group. Its value is just one-half the crosslinked interhead group center to center distance or 32.9 A. As noted above, the Hb ~8 dimer is itself an ellipsoid with radii of approximately 15 A x 20 A x 30 A. The measured value of 33 A for the distance of the crosslink from the center of the head group, and more particularly, the value of 29 A (half of 58 A, the inter-crosslinked a/? distance), indicate that the a/? dimer is lying with its major ellipsoid axis pointing approximately in the direction of the crosslink. This means that since the density of the cr/l dimer on the side of the crosslink, i.e. on the inside as defined in Figure 11, corresponds to the p chain with its N terminus on the extreme inside, then the density of the uj? dimer on the opposite side must be the a chain with its N terminus on the outside. Thus, approximate directions for two of the three axes of

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the L$ dimer ellipsoid have been specified by the electron micrograph images. The third axis, the 40 A one, must lie approximately perpendicular to the plane of the microscope grid running through the center of the a/3 dimer lobe. The cpangle in this case is the crosslink angle, cpc,(Figs 9(b) and 10). This value was determined to be 96.7 + 31.9 (Fig. 10(b)). The observed distribution of the (*rosslink angles is very broad. However, it is important to realize that the breadth of this distribution is due to the disparate values obtained for the two angles of some of the cross-linked pairs, as is shown in the plot (Fig. 10(a)). This disparity results from variation in the relative orientation of the molecular axes of the two crosslinked Cxl-1 molecules that are forced to lie in the same plane by attachment to the grid substrate. The relative orientation varies from approximately parallel to large divergence to convergence and even to crossing over (Figs 7 and 8). This is not surprising, because the chemical crosslink attaches a highly flexible /I N t)erminus to a very flexible lysine side-chain. In fact, the absolute difference between the two angles for a crosslinked pair of molecules (Fig. 10(a)) is a direct measure of the degree to which the two Cxl-1 molecular axes, as defined by the centers of the head groups, are not parallel. When the molecular axes are parallel, then the two measured crosslink angles should be the same and more accurately reflect the position of the crosslink. As the molecular axes become increasingly non-parallel, the values of the two angles diverge, but, on the average, they should vary about the same mean angle. Hence, despite the wide spread of angles observed for each crosslinked molecule pair, the mean value of the angle should accurately represent the position of the crosslink. The mean crosslink angle cpc= 96.7 and the radius p = 33 A define a circle at radius p, which intersects the surface of the head group of the Cxl-1 molecule upon which the /? N terminus must lie (Fig. 9(b)). To further specify the actual direction of the p N terminus on this circle, information must be obtained about the angle 9. If the Cxl-1 molecule head group were truly spherical, this would not be possible. However, the trans off-axis configuration of the Hb dimers on Cxl-1 provides a reference point for the definition of a datum value for 9, i.e. the x-axis. For each head group, the x-axis is defined as pointing in the direction in which t,he c$ dimer is off-axis, as shown in Figure 9(b). When the crosslinked Cxl-1 molecules were examined carefully, it was found that the overwhelming majority (16 out of 20) of the specimens were connected inside to inside (see Figs 7, 8 and 11). This indicates that the position of the crosslink and hence of the /? S terminus, lies at 9 = 0. A model of two crosslinked Cxl-1 molecules was constructed, placing the crosslink in the position (p, 8, cp)= (32.9 A, O, 96.7). This model is compared to electron micrograph images of two inside to inside crosslinked molecules that have quite different relative orientations of their Cxl-1 molecular axes (Fig. 13, rows 1 and 2). In Figure 13 (row 3), the flexibility about the crosslink has allowed the molecules to crossover. Nevertheless, the inside to inside configuration is clear. What can be said of the minority of crosslinked molecules, which appear to be linked differently than the above? Of the 20 molecules examined, three show an inside to outside crosslink that corresponds to 8 values of 0 and 180, respectively. Clearly, the /? N terminus cannot be at 9 = 0 in some molecules and

FIG.

13.

STRUCTURE

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HAPTOGLOBIN-HEMOGLOBIN

339

9 = 180 in others. One possible explanation for these molecules that is consistent with the previous designation of (p, 9, cp) = (32.9 A, 0, 96.7) is shown in Figure 13 (row 4). In this pair, one molecule of the pair has again landed normally, in a Z configuration, while the second has rolled over the first, adhering to the grid in the S configuration, showing an apparent attachment to its partner on the outside curve of its head group, even though the true connection is inside to inside. This roll-over conformation is presumably possible because of the considerable flexibility of the crosslink. However, it is less likely to occur than the more relaxed, simple inside to inside configuration, and thus it appears in only a small minority of cases. The last case, represented by only one of the 20 specimens, is outside to outside, and implies 9 = 180 in both molecules. This conformation is inconsistent with the co-ordinates assigned above. A closer look at this pair of molecules (Fig. 7: row 5, left) indicates that both molecules are eis rather than trans. In addition, the offaxis angles for their head groups are unusually high (Fig. 4(b); and see Discussion, section (b), above). Thus, this particular image is most likely to be the result of distortion of both of the Cxl-1 molecules as they fell down and adhered to the electron microscope grid. (d) Concluding remarks

Electron microscopy has provided an overall view of the Hpl-1 molecule and its interaction with Hb a/l dimers. A model for the structure is proposed that appears to account for most of the features observed. This model has the following properties. The general direction of the binding site of a/I on the H chain has been suggested. Using the schematic representation of the Cxl-1 molecule for reference (Fig. 2(b)), Cysl05H, which connects the H chain to the L chain lies just at the juncture of the spherical H chain lobe with the L chain filament (labeled point 3). The residues at cpO= 127, which are implicated in the Hb binding by biochemical experiments (Lustbader et al., 1983), specifically residues 130 to 137H, lie at the juncture between the two lobes of the ellipsoidal/bilobal head group. The orientation of the c$ dimer in the Cxl-1 molecule, as viewed in the electron microscope, has also been suggested. In Figure 2(b), the shortest axis of the a/I dimer would be aligned approximately in the direction of the line connecting the centers of the two head groups of the molecule. The aI/Iz interface, which is approximately perpendicular to the shortest axis of the dimer, would be pointing towards the H chain. The longest, axis of the a/? dimer would be pointing

Fm. 13. Micrographs of negatively stained crosslinked Cxl-1 dimers are compared to a bell and stick model of crosslinked Cxl-I dimer, which has been crosslinked at the position corresponding to (p: 8. cp) = (32.9 A, O, 96.7), i.e. in the inside to inside configuration. Row 1, the H-H axes of the 2 Cxl-1 molecules are approximately parallel; row 2, same as row 1, but with the H-H axes diverged from parallel; row 3 cross-over configuration, which is the result of the high flexihilitv about the crosslink; row 4, roll-over configuration, In this last case, the model still has the same inside to inside linkage. The lower Cxl-1 molecule lies in its usual Z configuration. The second molecule has rolled over the first almost 180 to an S-configuration, while still remaining crosslinked. In this roll-over configuration, the molecule appears to be inside to outside in projection.

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approximately perpendicular to the line that connects points 1 and 2 in Figure 2(b). The b N terminus lies on the inside and the 01 terminus is on the N outside (as defined in Fig. 11). Thus, an approximate relative orientation for the H chain and the Hb a/3 dimer has been derived by combining the results of the electron microscopy with biochemistry. Modelling studies using t#his starting conformation are currently underway. These studies are also being extended by locating other landmark sites on both Hb and Hp using macromolecular labeling, similar to the crosslinking studies described here. Recent efforts (Wail et al., 1982) indicate that small heavy-atom clusters will soon be available that will allow detailed labeling of specific residues and more accurate location of these sites on the molecule. Such studies will provide structural information at a considerably higher resolution than the more usual antibody labeling of protein assemblies,such as are typically performed on ribosomes (Lake, 1980) and other macromolecular aggregates. In the case of Cxl-1, it should allow more precise location of a variety of sites on the a/l dimer
sites will permit a more accurate determination of the relative orientation of the Hb afi dimer to the Hp H chain and hence provide new information about the binding site between these two molecules.
tram,

and on the H chain.

These

The relatively specific binding of Cxl-I to the electron microscope grid to give a off-axis, Z-configured view of the molecule suggests that, these micrograph
should be good candidates for the image processing techniques developed

images

by Joachim Frank and his co-workers for isolated molecules (Kessel et al., 1980; Frank et al., 1981). In view of the fact that the structure of Hb is known, that a good model is available for Hp H chain, and that a tentative relative orientation
for these two has been derived as described above, it will be interesting to see how

much structural information can be obtained and how high a resolution can be achieved from a systematic analysis of images of isolated molecules of this size, using these techniques.
We are grateful to Drs Reinhold and Ruth Benesch for the gift of purified crosslinked hemoglobin. We thank S. Berge Guiragossian, Kristin Chung and Frank Kito for skilled technical assistance. This research was supported by grants from the National Institutes of Health (HL-16601 to ,J.G.) and the Department of Energy (to J.S.W.). The Brookhaven STEM resource is supported by the National Institutes of Health Biotechnology Resources Branch grant RR-007 15. REFERENCES Arcoleo, J. P. & Greer, J. (1982).J. Biol. Chem.257, 10063-10068. Arnone, A., Benesch, R. E. & Benesch, R. (1977). .I. Mol. Biol. 115, 6277642. Baldwin, J. M. (1980). J. Mol. Biol. 136, 103-128. Benesch, R., Benesch, R. E., Yung, S. & Edalji, R. (1975). Biochem. Biophys. Res. Commun. 63, 1123-1129. Benesch, R. E., Ikeda, S. & Benesch, R. (1976). J. Biol. Chem. 251, 465-470. Bernini, L. F. & Borri-Voltattorni, C. (1970). Biochim. Biophys. Acta, 200, 203-219. Birktoft, J. J. & Blow, D. M. (1972). f. Mol. Biol. 68, 1877240. Black, J. A. & Dixon, G. H. (1968). Nature (London), 218, 736-741. Cohen, N. D., Due, J. A., Beegen, H. & Utter, M. F. (1979). J. Biol. Chem. 254, 9262-9269. Constans, J. & Viau, M. (1977). Amer. J. Hum. Genet. 29(3), 280-286. Eaton, J. W., Brandt, P., Mahoney, J. R. & Lee, J. T. (1982). Science, 215, 691-692.

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Estis. L. F. & Haschemeyer, R. H. (1980). Proc. Nut. Acad. Sci., U.S.A. 77. 3139-3143. Frank. J., Verschoor, A. & Boublik, M. (1981). Science, 214, 135331355. (ireer. J. (1980). Proc. Nut. A cud. Sci., U.S.A. 77, 3393-3397. (irerr, ,J. (1981). J. Mol. Biol. 153, 1027-1042. Haugen, T. H., Hanley, ,J. M. & Heath, E. C. (1981). J. Biol. Chem. 256, 1055-1057. Hwang, 1. K. & Greer, J. (1979). J. Biol. Chem. 254, 2265-2270. Hwang. P. K. & Greer, J. (1980). J. Biol. Chem. 255, 3038-3041. .Iavid. *J. (1978). (VW. Topics Haemat. 1, 151. 192. Kessel. M.. Frank, J. & Goldfarb, W. (1980). J. Supramol. Struct. 14. 405-422. Kurosky. A., Barnett, I). R., Rasco, M. A., Lee. T. & Bowman. B. H. (1974). Biochem. &net. 11. 279-293. Kurosky. A., Han-Hwa. K., Barnett, D. R., Rasco, M. A., Touchstone. B. & Bowman. 1%. H. (1975). Protides Biol. Fluids, Proc. Colloq. 22, 597-602. Kurosky. A., Hay, R. E., Kim, H.-H.. Touchstone, B., Rasco. M. & Bowman, B. H. (1976). Hiochemistry. 15, 5326-5336. Kurosky. A.. Barn&t, D. R., Lee, T.-H., Touchstone, B.. Hay, R. E., Amott, M. 8.. Bowman. B. H. & Fitch, W. M. (1980). Proc. Nat. Acad. Sci., C.I\.A. 77, 3388-3392. Ladner, R. C.. Heidner, E. J. & Perutz, M. F. (1977). J. Mol. Biol. 114. 385-414. Laemmli. U. K. (1970). Nature (London), 227, 680-685. Lake. J. A. (1980). In Ribosomes, Structure, Function an,d Genetics (Chambliss, G.. Craven, G. R.. Davies, K., Davis, J., Kahan. L. & Nomura, M., eds), pp. 671-691. Ilniversity Park Press. Baltimore. Laurel], (. B. (1959). Clin. Chim. Acta, 4, 79-81. Lust hader, .J. W.. Arcoleo, ,J. P., Birken. S. & Greer, ,J. (1983). J. Biol. Chem. 258, 12277 1234. Makinen, M. W., Milstien, J. B. & Kon, H. (1972). Biochemistry, 11, 3851-3860. Malchy. 13. & Dixon. G. H. (1973). Cunad. J. Biochem. 51, 249-264. Malchy, I~.. Rorstad, 0. & Dixon, G. H. (1973). Canad. J. Biochem. 51, 265-273. Mosesson, M. W.. Hainfeld, .J.. Wall, J. & Haschemeyer, R. H. (1981). J. Mol. Biol. 153, 695- 7 18. Nagrl. R. I,. & Gibson, Q. H. (1967). J. Biol. Chem. 242, 3428-3434. Ragel. IL. L. & Gibson, Q. H. (1971). J. Biol. Chem. 246, 69-73. Peacock. A. (.. Pastewka, ,J. V.. Reed, R. A. & Ness. A. T. (1970). Biochemistry. 9, 22752279.

Perutz. M. F. (1965). J. Mol. Biol. 13> 646-668. Pinura, ,I. (1971). Ser. Ha.emutol. 4, 1-183. Putnam, F. W. (1975). In The Plasma Proteins (Putnam. F. W.. ed.), vol. 2, pp. l-50. Academic Press, New York. Rafelson. M. E.. (loarrc. L., Moretti, .J. & Jayle, M. F. (1961). Nature (London), 191. 279~280. Sokal. R. R. & Rohlf. J. F. (1973). In Introduction to Riostatistics, pp. 1706175. W. H. Freeman and Company, San Francisco. Valrtte. I.. Waks. M., Wejman, J. C., Arcoleo, J. P. & Greer, J. (1981). J. Biol. (hem. 256, 672-679. Waks. M. & Alfsen, A. (1966). Arch. Biochem. Biophys. 113. 304-314. Waks. M.. Alfsen, A.. Schwaiger, S. & Mayer, 9. (1969). Arch. Biochem. Biophys. 132, 268% 278. Wall. J. (1979). In Introduction to Analytical Electron Microscopy (Hren, J. J., Goldstein. .I. I. & ,Joy. I). (1.. eds), pp. 333-342, Plenum Publishing Co., hew York. Wall. J.. Hainfeld. J., Boulett, P. A. & Singer, S. J. (1982). Ultramicroscopy, 8, 397-402. Woodcock. C. L. F.. Frado, L.-L. Y. & Wall, J. (1980). Proc. Nut. Acad. Sci., 7,.S.A. 77. 4818-4822. Yip, Y. K.. Waks, MM. & Beychok, S. (1972). J. Biol. Chem. 247. 7237-7244.
Edited by M. F. Moody