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An electron microscope study of the conformational change in bovine serum albumin at low pH
J. Mol. Biol. (1965) 14, 443-452
An Electron Microscope Study of the Conformational Change
in Bovine Serum Albumin at Low pH ELIZABETH MACLEAN SLAYTER
Department of Biology, Brandeis University Waltham, Mass., U.S.A. (Received 26 July 1965) Electron micrographs have been obtained of bovine serum albumin in solutions oflow ionic strength at pH values in the range 1·9 to 6·9. The predominant form of the molecule at neutral pH is found to be a globular particle approximately 60 A in diameter and 45 A high. Below pH 4, the molecule extends to a threadlike form which measures approximately 250 A long and 21 A in diameter at pH 1·9. While the details of the substructure of the acid form of serum albumin are below the resolution limit of the shadowing technique, micrographs indicate that extension must have occurred at several regions within the molecule. Compact doublet monomers are believed to be intermediates in the acid denaturation; similar structures also appear to be formed from native molecules as artifacts of specimen preparation. The proportions of native and denatured particle types found in micrographs of serum albumin are found to change with pH in the same manner as do hydrodynamic properties of the molecule. The configurational change is found to be reversible, and to be complete within 24 seconds after acidification of the albumin solution.
1. Introduction Bovine serum albumin and human serum albumin undergo a reversible conformational change when the pH is lowered below about 4·0. Physicochemical studies of the process by a number of workers, which have been reviewed by Foster (1960), show that at pH 4, native albumin takes up three hydrogen ions per molecule, and that extension ofthe molecule as the pH is further lowered is the result of mutual repulsion of positive changes. Foster suggested a model for the neutral and acid forms of serum albumin, according to which the molecule consists of four globular regions or "subunits", connected by fibrous regions. He suggested that above pH 4, close association of "subunits" would confer a singlet, globular structure on the molecule as a whole. At pH 4, halves of the molecule would separate to form a doublet structure upon uptake of hydrogen ions; at lower pH values the entire molecule would be extended. Luzzati, Witz & Nicolaieff (1961) have found by measurement of low-angle X-ray scattering that at pH 3·5, 37% of the BSAt molecule is converted from globular to extended form. The X-ray data do not indicate whether a single or several extended regions are formed. Recently, Chatterjee & Chatterjee (1965) have presented electron micrographs which suggest that the BSA molecule is of doublet form in neutral solution.
t
Abbreviation used: BSA, bovine serum albumin. 443
444
E. M. SLAYTER
In the present study, electron micrographs of BSA have been obtained at a series of pH values in the range 1·9 to 6'9, in order to determine what changes occur in over-all molecular shape as a result of the acid-induced transition in molecular structure. The acid denaturation of albumin furthermore provided an opportunity for correlation of electron microscope observations with events which have been quite thoroughly characterized in solution.
2. Materials and Methods (a) M aterials The BSA used was a crystalline preparation obtained from the Sigma Chemical Co. All preparations were dialyzed for at least 3 days against frequent changes of glassdistilled water. BSA solutions for electron microscopy were unbuffered, adjustment of pH being achieved by additions of acetic acid, hydrochloric acid or ammonium hydroxide. Unbuffered solutions were used primarily because the extent of conformational change in BSA is known to be a maximum at low ionic strength (Yang & Foster, 1954; Reichman & Charlwood, 1954). The use of solutions of low ionic strength is also desirable because the dimerization of albumin on standing at low pH is thus minimized (Foster, 1960, p. 216) and because background impurities in electron micrographs are reduced. The latter is an important consideration in the microscopy of particles such as the BSA molecule, which are of marginal size for observation in electron micrographs. (b) Electron micr08copy
BSA solutions at a concentration of about 0·1 rogfml. were sprayed on freshly cleaved mica and shadowed with platinum at an angle of 10: 1 as described by Hall (1956). Micrographs were obtained on an RCA EMU-3-G electron microscope at an instrumental magnification of about 16,000. Prints for measurement or counting of particle types were prepared at total magnifications of about 120,000. Measurements were roade of both particle diameters and shadow lengths, using a 60 A correction for the accumulation of shadowing metal (Hall, 1960). (c) Oounts of partide types Areas for counting of particle types were selected only by choosing areas of appropriate densityof particles. At least three separate specimen grids were photographed for counting at each pH. Since individual BSA molecules are visible on the electron microscope screen only with some difficulty, it is reasonable to conclude that the areas selected for counting were indeed random. At least 6 plates were enlarged for counting at each pH. Particles were classified as one of 5 types, as explained further under Results. These were globular, aggregate, 8emi-native, threadlike, and unclassified. The "unclassified" small particles, representing 10% or less of the total count, were considered to be breakdown products or impurities, and were not included in percentage totals. Particle counts were made independently by several observers to whom the pH values of the preparations counted were unknown. (d) Reversibility of the conformational change A preparation which was observed in the electron microscope to be of typical "aciddenatured" appearance at pH 3·3 was restored to neutral pH by exhaustive dialysis against distilled water.
(e) Time course of the conformational change In order to determine the speed of the conformational change as observed by electron microscopy, two experiments were performed in which preparations were sprayed at intervals following addition of acid. In one experiment, specimens were prepared 26 sec, 5 min and 15 min following acidification to pH 2'8; in the other, 24 sec, 51 sec, 2 min and 4 min following acidification to pH 3·1.
CONFORMATIONAL CHANGE OF SERUM ALBUMIN
445
3. Results (a) Native and denatured BSA
Electron micrographs of BSA prepared from distilled water are generally inhomogeneous. The pH of such preparations is in the range 5·5 to 6,0, which is close to the isoelectric pH of 5·3 at zero ionic strength (Foster, 1960, p. 194). Aggregation of BSA molecules is expected in this pH range. Nevertheless, approximately spherical particles can be selected from micrographs of BSA prepared from distilled water which are of appropriate dimensions to correspond with the established molecular weight of 66,000 (Schultze, Heimburger & Frank, 1962). An increased proportion of these particles is observed when the pH is maintained somewhat further from the isoelectric point. Plate I(a) shows a BSA preparation maintained at pH 6·9 by addition of a trace of ammonia; a few typical globular BSA molecules are circled. Doublets were also observed at pH 6'9, but less frequently than the singlet globular particles; a group of doublets is illustrated in Plate I(b). Plate II shows a BSA preparation sprayed from dilute hydrochloric acid solution at pH 1·9. Although occasional "native" molecules are seen, the predominant molecular species is a short thread. Sometimes the threads are folded over upon themselves; more typically they are extended. The threads as a group seem to be more or less uniform along their length; there is no evidence for a predominant "ball and chain" structure. Measurements were made of several hundred particles of both types. "Globular" particles were measured at pH 6·9 and 4,2, whereas "threadlike" particles were measured at pH 4,2, 3·3 and 1·9. The average corrected diameters and heights are given in Table 1, together with the molecular weights calculated thereform. These measurements establish the identity of the "globular" particles selected at pH 6·9 and 4·2. The molecular weights of "threadlike" particles are identical at pH 4·2 and 1·9; the value obtained at pH 3·3 is probably within experimental error. "Threadlike" particles are most fully extended at pH 1·9. The distributions of dimensions of the two particle types in the preparation at pH 4·2 is illustrated in Fig. 1. The reversibility of the acid-induced conformational change has been confirmed by electron microscopy.
TABLE
1
Measurements of native and denatured BSA Particle type
pH
Diametert (A)
Height
Native
6·9 4·2 4·2 3·3 1·9
56 60 170 136 250
46 45 25 23 21
Denatured
(A)
Assumed shape M (calc.):!:
Oblate ellipsoid Cylindrical rod
61,500 69,500 68,500 46,400 71,000
t Corrected for 60 A accumulation of shadowing metal. ~ M = 4/31Tab2 d N for oblate ellipsoid and M = 1TT2 l d N for cylindrical rod; d
=
1·36 glee.
E. M. SLAYTER
446
50 on OJ
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lA) FIG . 1. Dimensions of "glob ular" and " t hreadlike" particles at pH 4·2. (a) Particle diamete rs, corrected for 60 A of shadowing me t al. (b) Particle h eigh ts.
Upon addition of acid to a neutral BSA solut ion, alteration of molecular structure appears to be complete within 24 seconds, the minimum time required for specimen preparation. (b) Pa rticle type counts as a f unction of pH
F igure 2, reproduced from H arrington, Johnson & Ottewill (1956), illustrates the sedimentat ion coefficient, diffusion coefficient, intrinsic viscosity and polarization of fluorescence by conjugates of BSA as a fun ction of pH. To t he exte nt that the BSA molecules seen in elect ron micrographs are not distorted by artifacts, a graph of the proportions of native or denatured particles counted in micrographs, as a function of pH, should be a curve similar in shape to those shown in Fig. 2. Particle types were counted in preparations sprayed from the following solutions:
pH 6·9 5·8 4·2 3·8 3·4 3·1 1·9
Solvent Dilute ammonia Distilled water Dilute hydrochloric Dilute hydrochloric Dilute hydrochloric Dilute acetic acid Dilute hydrochloric
acid acid acid acid.
Not all particles could be classified as "globular" or "t hreadlike"-Le., "native" and "denatured", respecti vely. Some larger particles, cast ing relat ively long shadows, appeared to be aggregates of na ti ve molecules, while a larger group of "semi-nat ive"
CONFORMATIONAL CHANGE OF SERU :&-f ALBUMIN
447
s."m'."'", J'
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Diffusion I
----... --
I
I
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Polarizat ion
Intrinsic viscosity
Fro, 2. Sedimentation coefficient, diffusion coefficient, polarization of fluorescence of conjugates and intrinsic viscosity of BSA as a function of pH. (After Harrington et al., 1956.)
particles included the doublet type of structure illustrated in Plate I(b) and also a less well-defined group of particles which lacked the spherical character and relatively long shadows of "globular" particles without being clearly threadlike. " Globular " and " aggregate" particles were considered to be native material; "semi-native" and "threadlike" particles; which appeared to have undergone at least the initial stages of extension, were collectively classified as denatured. .Typical examples of each of the four particle types are illustrated in Plate III. Typical fields from the preparations counted at pH 4,2, 3·8 and 1·9 are shown in Plate IV. The pooled results of counts of particle types by three observers are given in Tables 2 and 3, and Figs 3 and 4. Table 2 and Fig. 3 present the results directly as obtained, whereas in Table 3 and Fig. 4 they have been corrected to account for the fact that each particle scored as an aggregate represents more than one BSA molecule . Polymeric forms of BSA are principally dimers, but an assortment of higher multiples may also occur (Hartley, Peterson & Sober, 1962); it was therefore assumed that, on the average, each aggregate represents 2·5 monomers. The effect of the correction is to flatten the curve given in Fig. 4 in the pH region 4 to 7, resulting in a plot surprisingly similar in form to those of parameters measured in solution as shown in Fig. 2. Despite the relatively low proportion (about 50%) of native molecules observed in neutral solutions (see Discussion), it would appear that alterations of the appearance of the group of molecules as a whole, in electron micrographs, reflect events as they occur in solution. An exception to the above is observed in certain preparations made at pH 3·5 or lower. A series of counts of particle types carried out at low pH is described in Table 4 and Fig. 5. Figure 5(a) is a plot of counts made from preparations in hydrochloric acid solution; Fig. 5(b) shows the results obtained from acetic acid solution. The proportions of particle types observed are solvent-dependent below about pH 3,5, those from acetic acid solution being anomalous with respect to the hydrodynamic data.
448
E. M. SLAYTER TABLE
2
Counts of particle types on BSA micrographs (I)
(2)
(3)
(4)
(5)
(6)
%
%
%
%
%
(7)
%
pH
"Globular"
"Aggregates"
"Semi-native'
"Threadlike"
Nativet
Denatured:j:
6·9 5·8 4·2 3·8 3·4 3·1 1·9
38·5 30·0 18·5 13·5 13·0 9·5 4·5
6·0 8·0 16·5 5·5 4·5 2·0 0·5
38·0 38·0 48·0 39·0 32·5 19·0 25·0
17·5 24·0 17·0 42·0 50·0 69·5 70·0
44·5 38·0 35·0 19·0 17·5 II·5 5·0
55·5 62·0 65·0 81·0 82·5 88·5 95·0
t Sum of columns (2) and (3). :j: Sum of columns (4) and (5).
TABLE
3
Counts of types of particles on BSA micrographs, corrected by the assumption that one "aggregate" represents, on the average, 2·5 "globular" particles (I)
(2)
(3)
(4)
(5)
(6)
%
%
%
%
%
(7)
%
pH
"Globular"
"Aggregated"
"Semi-native"
"Threadlike"
Nativet
Denaturedj
6·9 5·8 4·2 3·8 3-4 3·1 1·9
35·5 27·0 14·5 12·5 12·5 9·5 4·5
13·0 18·0 33·5 12·5 10·5 4·5 2·0
35·0 33·5 38·5 36·0 30·5 18·5 25·0
16·0 21·5 13·5 39·0 46·5 67·5 68·5
48·5 45·0 48·0 25·0 23·0 14·0 6·5
51·5 55·0 52·0 75·0 77·0 86·0 93·5
t Sum of columns (2) and (3). t Sum of columns (4) and (5).
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pH
FIG. 3
FIG. 4
FIG. 3. Proportion of native particle types ("globular" and "aggregate") in electron micrographs of BSA as a function of pH. Uncorrected observations. FIG. 4. Proportion of native molecules ("globular" and "aggregated") in electron micrographs of BSA as a function of pH. Observations corrected by the assumption that one "aggregate" represents, on the average, 2·5 "globular" particles.
PLATE 1. Bovine serum albumin (pH 6·9). (b) Doublet particles.
X
200,000. (a) Typical singlet globular particles.
[facing p. 448
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II. Bovine serum albumin, pH 1·9.
X
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PLATE III. Exam pl es of types of bovine se r u m a lb umin parti cle. (b ) " a ggrega t e" ; (e) "semi-native " ; (d) " t h rea d lik e" .
X
160 ,000. (a) "Globu lar";
PLATE
IV. Bovine serum albumin.
X
160,000. (a) pH 4·2, (b) pH 3·8, (c) pH Hl.
CONFORMA TIONAL CHANGE OF SERUM ALBUMIN TABLE
449
4
Oounts of particle types of B S A at low pH values
%
%
%
%
%
%
pH
Solvent
"Globular"
"Aggregated"
"Semi. native"
"Thread. like"
Native
Denature d
1'9 2·8 3·06 3·12 3·3 3'4 3·6 4·1 4·2
HCl HAc HAc HAc HAc Hel HAc HAc HCl
4·5 20·0 8·0 9·5 2·0 12'5 6·5 34·0 14·5
2·0 17·0 8·0 4:5 4·5 10·5 7·5 19·0 33·5
25·0 1l·0 30·0 18'5 14· 0 30·5 30·0 14·0 38· 5
68·5 52·0 54·0 67·5 79·5 46'5 56·0 33·0 13'5
6·5 37·0 16·0 14·0 6·5 23·0 14·0 53·0 48·0
93·5 63·0 84·0 86·0 93·5 77-0 86·0 47·0 52·0
(Corrected on the assumption that one "Aggregate " represents, on the average, 2·5 "G lobular" particles.)
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50
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FIG. 5. Proportions of native molecules ("globular" and "aggregated") in electron micrographs of BSA. Observations corrected on the assumption that one "aggregate" represents, on the average, 2'5 " globular " particles. (a) Preparations from hydrochloric acid solutions. (b) Preparations from acetie acid solutions.
4. Discussion Measurements of particle sizes from shadowed electron micrographs are subject t o unccrtainty in the choice of correction for the accumulation of sha dowing metal; since particle volumes and molecular weights are functions of the cube of linear dimensions, molecular weights obtained from electron microscopy for particles as small as the BSA molecule must be accepted as estimates reliable only to about ± 50%. Attempts to avoid these uncertainties by the use of negative-contrast methods have been unsuccessful (Valentine, 1959). Nevertheless, macromolecules in the 50 to 60 A size range may be studied in shadowed electron micrographs, since it is possible thereby to establish whether particle sizes are in a range consistent with molecular weights as determined by physicochemical methods, and, particularly, to observe directly the over -all shape of particles down to the 20 or 30 A limit imposed by the grain of shadowing metal. 29
450
E. M. SLAYTER
In the present study it has been possible to identify particles of average height 45 A and average diameter 60 A as a major component in electron micrographs of BSA prepared from solutions in the pH range from 4·2 to 6·9. Below pH 4,2, these globular particles are replaced by threadlike particles which, at pH 1,9, have an average diameter of 21 A and average length 250 A. Both of these sets of dimensions are consistent with the established molecular weight of BSA, namely, 66,000. Furthermore, as may be seen from comparison of Figs 4 and 2, the extent of the transition from native to "denatured" BSA observed in electron micrographs, as a function of pH, rather closely parallels the alterations of properties measured in solution. It is encouraging to find that the electron micrographs represent the expected behavior of the molecules as deduced from very extensive physicochemical studies. This result suggests that artifacts of specimen preparation for electron microscopy-including collapse upon dehydration, effects of sudden changes of pH, and disruption by surface tension forces during drying down-need not necessarily preclude the use of micrographs for the interpretation of events as they occur in solution. Nevertheless, certain questions arise concerning the observations described here. It is necessary to explain why anomalous proportions of particle types are observed in preparations made from acetic acid solutions below pH 3·5. The extent to which possible structures of the acid form of albumin can be distinguished from electron micrographs must be considered. The fact that no more than 50% of native particle types are observed in electron micrographs at neutral pH must be accounted for. Finally, the relation of the doublet molecules described by Chatterjee & Chatterjee (1965) to the approximately spherical monomer' found here should be clarified. The apparent re-appearance of native particle types in acetic acid solutions below pH 3·5 may be due to the lowered surface tension of that solvent. As shown in Fig. 6, the effect of HCI upon solvent surface tension is virtually negligible at the highest
(a)
73r7"",-------...,
60~-........--...L.-.~---'
o
4
Weight % acetic acid
72~_--L.._ _':-_....J
o
4
Weight % of Hel
FIG. 6. Surface tension (y in dynes/cm-) of solvent solutions. Arrows indicate maximum weight per cent of each substance employed. Data plotted from Handbook of Ohemistr'lJ and Physics. 45th Ed. 1965, P- F·16. (a) Acetic acid solutions, 30°0. (b) Hydrochloric acid solutions, 20°0,
concentrations used in this study, whereas the highest concentration of acetic acid used was sufficient to lower solvent surface tension by about 7%. The acid form of BSA may no longer be constrained to dry down in extended configuration from solutions of lowered surface tension. The results of Luzzati et al. (1961) indicated a 37% conversion ofBSA from globular
CONFORMATIONAL CH A N G E OF SERUM ALB UMIN
451
to fibrou s form at pH 3·5. It is of interest to consider whether p articles with one or severa l fibrou s regions could be dist inguished from each other, and from native E SA, in electron micrographs. If it were exactly spher ical, t he nati ve E SA molecule would be 54 A in diamet er. If 63 % of the molecule remai ns as a single globular region after a cid-denaturation , t h is port ion would have a molecular weight of 41,500, corresponding to a sphe re 45 A in dia mete r . Since measurements of particle diameters ar e uncertain to about 10 A, the globular portion of the denatured molecule would be essentially indist inguishable from t he original nat ive form. If the fibrous porti on of t he molecule were only partially uncoiled , it might measure about 20 A X 100 A, so that the denatured molecule would appear in micrographs as a " ball and cha in" struct ure. If the fibrous portion of the molecule were a full y extended polypeptide cha in, it would measure less than 10 A in diamet er and might not be det ected. Neither of these possibiliti es is consiste nt with the observat ions. If, alternatively, the acid form of ESA has a structure such as suggested by F oster, in which four globular regions are separated by three fibrous regions, the individual globular regions would have an average molecular weight of 10,500, corresponding to a diameter of 28 A. For incompletely extended fibrous regions, the differen ce in diam et er between "globular " and " fibrous" regions would thus be reduced t o ab out 10 A, and, after coating wit h sha dowing metal, such a molecule would be expected to appear as a more or less uniform t hread. If the fibrou s regions were fully extended polyp eptide chains, the molecule should appear as a series of fairly well-separated spheres ra ther than the threadlike stru ct ure observe d. It is possible, however , that the fibrou s regions might be fully extende d in solution, but par tially contract on dr ying. Such a possibility serves to reconcile the X-ray dat a of Lu zzati et al. (1961) with t he ap pearance of the acid form in micrographs. In summary, while electron micrographs provide no ba sis for suggest ing that there ar e, specifically, four "globul ar " regions in the acid form of ESA, the obser vation of t hreadlike particles in E SA solut ions at low pH indicat es that exte nsion occurs at several points in the molecule ra t her than in a single region. Th e observation, at neutral pH, of occasional doublet st ruct ures and of not more than 50% of native par ticles may be exp lained by t he following hypotheses. ES A exists in solut ion ent irely as t he native configurat ion above pH 4·5 . As shown by titrati on studies (F oster, 1960, p . 212), three hydrogen ions are t aken up per molecule over a narrow range of pH at about pH 4·0. This causes partial loosening of t ertiary str ucture, forming a doublet molecule. The same reaction may rather easily be mimi cked on drying down, by the action of surface t ension or other forces. The doublet molecule is thus considered to represent the expandible form of ESA deduced by Tanford (1961) and Foster (1960). The doublet "subunits" mu st be intermeshed when the molecule is in its fully native form , producing an approximately spherical shape of the latter. The observation of some " t hreadlike " particles in preparations at pH 6·0 indi cates that not only doubl ets but fully extended configurations are form ed from nati ve E SA as an artifact of drying. Table 2 shows t hat, at pH 4,2, 5·8 a nd 6,9, approximately 50 % of molecules ar e observed in micrographs as " nat ive" E SA, 35 % as " semi-native" (including doublets) and 15% as "threadlike". If one accordingly assumes that , at any pH, 50% of native molecules will be converted on dr ying t o the "semi-na t ive" form , and also that 30% of "s emi-native" par ticles, both pre-exist ing and newly formed , will at the sam e tim e be conver ted to the " t hreadlike " form , it is possible to est ima te t he proportions of
452
E. M. SLAYTER
particles present in solution from those observed in electron micrographs. The following equations apply: (1) N; = 2No
Ss =
So -0,7 No --=---,-------=0·7
T s = To -3/7 So
(2) (3)
in which No, So and To are proportions of native "semi-native" and "threadlike" molecules counted in electron micrographs, and N s, Ss and T s are the proportions of the corresponding particle types in solution. Application of this treatment to the results in Table 3 yields Fig. 7, which illustrates the estimated proportion of "doublets" ("semi-native" particles) in solution. Figure 7, in excellent agreement with
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Q.
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357 pH
FIG. 7. Estimated proportions of doublet ("semi-native") particles in solution as a function of pH.
the predictions of physicochemical studies except for an anomalous value at pH 1,9, illustrates the sudden appearance of a population of "semi-native" molecules at pH 4, and its more gradual decay as the pH is lowered. I thank Miss F. Cotton and Mrs L. Dow for technical assistance, and Mrs M. Federbush and Dr A. D. Dingle for counts of particle types. This work was supported by a grant (GM-09959) from the National Institutes of Health. REFERENCES Chatterjee, A. & Chatterjee, S. N. (1965). J. Mol. Biol. 11, 432. Foster, J. F. (1960). In The Plasma Proteins, vol. 1, ed. by F. W. Putnam, p. 206. New York & London: Academic Press. Hall, C. E. (1956). J. BiophY8. Biochem. Oytol. 2, 625. Hall, C. E. (1960). J. Biophy8. Biochem. Oytol. 7, 613. Harrington, W. F., Johnson, P. & Ottewill, R. H. (1956). Biochem. J. 62, 569. Hartley, R. W., Jr., Peterson, E. A. & Sober, H. A. (1962). Biochemi8try, 1, 60. Luzzati, V., Witz, J. & Nicolaieff, A. (1961). J. Mol. Biol. 3, 379. Reichmann, M. & Charlwood, P. (1954). Oanad. J. Ohem, 32, 1092. Schultze, H. E., Heimburger, N. & Frank, G. (1962). Biochem. Z. 336, 388. Tanford, C. (1961). Phqsical Chemistrs] oj Macromolecules, New York & London: John Wiley & Sons. Valentine, R. C. (1959). Nature, 184, 1838. Yang, J. T. & Foster, J. F. (1954). J. Amer. Ohern, Soc. 76, 1588.
An Electron Microscope Study of the Conformational Change
in Bovine Serum Albumin at Low pH ELIZABETH MACLEAN SLAYTER
Department of Biology, Brandeis University Waltham, Mass., U.S.A. (Received 26 July 1965) Electron micrographs have been obtained of bovine serum albumin in solutions oflow ionic strength at pH values in the range 1·9 to 6·9. The predominant form of the molecule at neutral pH is found to be a globular particle approximately 60 A in diameter and 45 A high. Below pH 4, the molecule extends to a threadlike form which measures approximately 250 A long and 21 A in diameter at pH 1·9. While the details of the substructure of the acid form of serum albumin are below the resolution limit of the shadowing technique, micrographs indicate that extension must have occurred at several regions within the molecule. Compact doublet monomers are believed to be intermediates in the acid denaturation; similar structures also appear to be formed from native molecules as artifacts of specimen preparation. The proportions of native and denatured particle types found in micrographs of serum albumin are found to change with pH in the same manner as do hydrodynamic properties of the molecule. The configurational change is found to be reversible, and to be complete within 24 seconds after acidification of the albumin solution.
1. Introduction Bovine serum albumin and human serum albumin undergo a reversible conformational change when the pH is lowered below about 4·0. Physicochemical studies of the process by a number of workers, which have been reviewed by Foster (1960), show that at pH 4, native albumin takes up three hydrogen ions per molecule, and that extension ofthe molecule as the pH is further lowered is the result of mutual repulsion of positive changes. Foster suggested a model for the neutral and acid forms of serum albumin, according to which the molecule consists of four globular regions or "subunits", connected by fibrous regions. He suggested that above pH 4, close association of "subunits" would confer a singlet, globular structure on the molecule as a whole. At pH 4, halves of the molecule would separate to form a doublet structure upon uptake of hydrogen ions; at lower pH values the entire molecule would be extended. Luzzati, Witz & Nicolaieff (1961) have found by measurement of low-angle X-ray scattering that at pH 3·5, 37% of the BSAt molecule is converted from globular to extended form. The X-ray data do not indicate whether a single or several extended regions are formed. Recently, Chatterjee & Chatterjee (1965) have presented electron micrographs which suggest that the BSA molecule is of doublet form in neutral solution.
t
Abbreviation used: BSA, bovine serum albumin. 443
444
E. M. SLAYTER
In the present study, electron micrographs of BSA have been obtained at a series of pH values in the range 1·9 to 6'9, in order to determine what changes occur in over-all molecular shape as a result of the acid-induced transition in molecular structure. The acid denaturation of albumin furthermore provided an opportunity for correlation of electron microscope observations with events which have been quite thoroughly characterized in solution.
2. Materials and Methods (a) M aterials The BSA used was a crystalline preparation obtained from the Sigma Chemical Co. All preparations were dialyzed for at least 3 days against frequent changes of glassdistilled water. BSA solutions for electron microscopy were unbuffered, adjustment of pH being achieved by additions of acetic acid, hydrochloric acid or ammonium hydroxide. Unbuffered solutions were used primarily because the extent of conformational change in BSA is known to be a maximum at low ionic strength (Yang & Foster, 1954; Reichman & Charlwood, 1954). The use of solutions of low ionic strength is also desirable because the dimerization of albumin on standing at low pH is thus minimized (Foster, 1960, p. 216) and because background impurities in electron micrographs are reduced. The latter is an important consideration in the microscopy of particles such as the BSA molecule, which are of marginal size for observation in electron micrographs. (b) Electron micr08copy
BSA solutions at a concentration of about 0·1 rogfml. were sprayed on freshly cleaved mica and shadowed with platinum at an angle of 10: 1 as described by Hall (1956). Micrographs were obtained on an RCA EMU-3-G electron microscope at an instrumental magnification of about 16,000. Prints for measurement or counting of particle types were prepared at total magnifications of about 120,000. Measurements were roade of both particle diameters and shadow lengths, using a 60 A correction for the accumulation of shadowing metal (Hall, 1960). (c) Oounts of partide types Areas for counting of particle types were selected only by choosing areas of appropriate densityof particles. At least three separate specimen grids were photographed for counting at each pH. Since individual BSA molecules are visible on the electron microscope screen only with some difficulty, it is reasonable to conclude that the areas selected for counting were indeed random. At least 6 plates were enlarged for counting at each pH. Particles were classified as one of 5 types, as explained further under Results. These were globular, aggregate, 8emi-native, threadlike, and unclassified. The "unclassified" small particles, representing 10% or less of the total count, were considered to be breakdown products or impurities, and were not included in percentage totals. Particle counts were made independently by several observers to whom the pH values of the preparations counted were unknown. (d) Reversibility of the conformational change A preparation which was observed in the electron microscope to be of typical "aciddenatured" appearance at pH 3·3 was restored to neutral pH by exhaustive dialysis against distilled water.
(e) Time course of the conformational change In order to determine the speed of the conformational change as observed by electron microscopy, two experiments were performed in which preparations were sprayed at intervals following addition of acid. In one experiment, specimens were prepared 26 sec, 5 min and 15 min following acidification to pH 2'8; in the other, 24 sec, 51 sec, 2 min and 4 min following acidification to pH 3·1.
CONFORMATIONAL CHANGE OF SERUM ALBUMIN
445
3. Results (a) Native and denatured BSA
Electron micrographs of BSA prepared from distilled water are generally inhomogeneous. The pH of such preparations is in the range 5·5 to 6,0, which is close to the isoelectric pH of 5·3 at zero ionic strength (Foster, 1960, p. 194). Aggregation of BSA molecules is expected in this pH range. Nevertheless, approximately spherical particles can be selected from micrographs of BSA prepared from distilled water which are of appropriate dimensions to correspond with the established molecular weight of 66,000 (Schultze, Heimburger & Frank, 1962). An increased proportion of these particles is observed when the pH is maintained somewhat further from the isoelectric point. Plate I(a) shows a BSA preparation maintained at pH 6·9 by addition of a trace of ammonia; a few typical globular BSA molecules are circled. Doublets were also observed at pH 6'9, but less frequently than the singlet globular particles; a group of doublets is illustrated in Plate I(b). Plate II shows a BSA preparation sprayed from dilute hydrochloric acid solution at pH 1·9. Although occasional "native" molecules are seen, the predominant molecular species is a short thread. Sometimes the threads are folded over upon themselves; more typically they are extended. The threads as a group seem to be more or less uniform along their length; there is no evidence for a predominant "ball and chain" structure. Measurements were made of several hundred particles of both types. "Globular" particles were measured at pH 6·9 and 4,2, whereas "threadlike" particles were measured at pH 4,2, 3·3 and 1·9. The average corrected diameters and heights are given in Table 1, together with the molecular weights calculated thereform. These measurements establish the identity of the "globular" particles selected at pH 6·9 and 4·2. The molecular weights of "threadlike" particles are identical at pH 4·2 and 1·9; the value obtained at pH 3·3 is probably within experimental error. "Threadlike" particles are most fully extended at pH 1·9. The distributions of dimensions of the two particle types in the preparation at pH 4·2 is illustrated in Fig. 1. The reversibility of the acid-induced conformational change has been confirmed by electron microscopy.
TABLE
1
Measurements of native and denatured BSA Particle type
pH
Diametert (A)
Height
Native
6·9 4·2 4·2 3·3 1·9
56 60 170 136 250
46 45 25 23 21
Denatured
(A)
Assumed shape M (calc.):!:
Oblate ellipsoid Cylindrical rod
61,500 69,500 68,500 46,400 71,000
t Corrected for 60 A accumulation of shadowing metal. ~ M = 4/31Tab2 d N for oblate ellipsoid and M = 1TT2 l d N for cylindrical rod; d
=
1·36 glee.
E. M. SLAYTER
446
50 on OJ
.~ o
a.
'0 ~
"' OJ
v "-2o
25
a.
'0 ~
lA) FIG . 1. Dimensions of "glob ular" and " t hreadlike" particles at pH 4·2. (a) Particle diamete rs, corrected for 60 A of shadowing me t al. (b) Particle h eigh ts.
Upon addition of acid to a neutral BSA solut ion, alteration of molecular structure appears to be complete within 24 seconds, the minimum time required for specimen preparation. (b) Pa rticle type counts as a f unction of pH
F igure 2, reproduced from H arrington, Johnson & Ottewill (1956), illustrates the sedimentat ion coefficient, diffusion coefficient, intrinsic viscosity and polarization of fluorescence by conjugates of BSA as a fun ction of pH. To t he exte nt that the BSA molecules seen in elect ron micrographs are not distorted by artifacts, a graph of the proportions of native or denatured particles counted in micrographs, as a function of pH, should be a curve similar in shape to those shown in Fig. 2. Particle types were counted in preparations sprayed from the following solutions:
pH 6·9 5·8 4·2 3·8 3·4 3·1 1·9
Solvent Dilute ammonia Distilled water Dilute hydrochloric Dilute hydrochloric Dilute hydrochloric Dilute acetic acid Dilute hydrochloric
acid acid acid acid.
Not all particles could be classified as "globular" or "t hreadlike"-Le., "native" and "denatured", respecti vely. Some larger particles, cast ing relat ively long shadows, appeared to be aggregates of na ti ve molecules, while a larger group of "semi-nat ive"
CONFORMATIONAL CHANGE OF SERU :&-f ALBUMIN
447
s."m'."'", J'
~
'" :.
I
Diffusion I
----... --
I
I
~/
Polarizat ion
Intrinsic viscosity
Fro, 2. Sedimentation coefficient, diffusion coefficient, polarization of fluorescence of conjugates and intrinsic viscosity of BSA as a function of pH. (After Harrington et al., 1956.)
particles included the doublet type of structure illustrated in Plate I(b) and also a less well-defined group of particles which lacked the spherical character and relatively long shadows of "globular" particles without being clearly threadlike. " Globular " and " aggregate" particles were considered to be native material; "semi-native" and "threadlike" particles; which appeared to have undergone at least the initial stages of extension, were collectively classified as denatured. .Typical examples of each of the four particle types are illustrated in Plate III. Typical fields from the preparations counted at pH 4,2, 3·8 and 1·9 are shown in Plate IV. The pooled results of counts of particle types by three observers are given in Tables 2 and 3, and Figs 3 and 4. Table 2 and Fig. 3 present the results directly as obtained, whereas in Table 3 and Fig. 4 they have been corrected to account for the fact that each particle scored as an aggregate represents more than one BSA molecule . Polymeric forms of BSA are principally dimers, but an assortment of higher multiples may also occur (Hartley, Peterson & Sober, 1962); it was therefore assumed that, on the average, each aggregate represents 2·5 monomers. The effect of the correction is to flatten the curve given in Fig. 4 in the pH region 4 to 7, resulting in a plot surprisingly similar in form to those of parameters measured in solution as shown in Fig. 2. Despite the relatively low proportion (about 50%) of native molecules observed in neutral solutions (see Discussion), it would appear that alterations of the appearance of the group of molecules as a whole, in electron micrographs, reflect events as they occur in solution. An exception to the above is observed in certain preparations made at pH 3·5 or lower. A series of counts of particle types carried out at low pH is described in Table 4 and Fig. 5. Figure 5(a) is a plot of counts made from preparations in hydrochloric acid solution; Fig. 5(b) shows the results obtained from acetic acid solution. The proportions of particle types observed are solvent-dependent below about pH 3,5, those from acetic acid solution being anomalous with respect to the hydrodynamic data.
448
E. M. SLAYTER TABLE
2
Counts of particle types on BSA micrographs (I)
(2)
(3)
(4)
(5)
(6)
%
%
%
%
%
(7)
%
pH
"Globular"
"Aggregates"
"Semi-native'
"Threadlike"
Nativet
Denatured:j:
6·9 5·8 4·2 3·8 3·4 3·1 1·9
38·5 30·0 18·5 13·5 13·0 9·5 4·5
6·0 8·0 16·5 5·5 4·5 2·0 0·5
38·0 38·0 48·0 39·0 32·5 19·0 25·0
17·5 24·0 17·0 42·0 50·0 69·5 70·0
44·5 38·0 35·0 19·0 17·5 II·5 5·0
55·5 62·0 65·0 81·0 82·5 88·5 95·0
t Sum of columns (2) and (3). :j: Sum of columns (4) and (5).
TABLE
3
Counts of types of particles on BSA micrographs, corrected by the assumption that one "aggregate" represents, on the average, 2·5 "globular" particles (I)
(2)
(3)
(4)
(5)
(6)
%
%
%
%
%
(7)
%
pH
"Globular"
"Aggregated"
"Semi-native"
"Threadlike"
Nativet
Denaturedj
6·9 5·8 4·2 3·8 3-4 3·1 1·9
35·5 27·0 14·5 12·5 12·5 9·5 4·5
13·0 18·0 33·5 12·5 10·5 4·5 2·0
35·0 33·5 38·5 36·0 30·5 18·5 25·0
16·0 21·5 13·5 39·0 46·5 67·5 68·5
48·5 45·0 48·0 25·0 23·0 14·0 6·5
51·5 55·0 52·0 75·0 77·0 86·0 93·5
t Sum of columns (2) and (3). t Sum of columns (4) and (5).
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-"'v '-2 a
0-
25
0)
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a Z ~
1
3
5
7
pH
FIG. 3
FIG. 4
FIG. 3. Proportion of native particle types ("globular" and "aggregate") in electron micrographs of BSA as a function of pH. Uncorrected observations. FIG. 4. Proportion of native molecules ("globular" and "aggregated") in electron micrographs of BSA as a function of pH. Observations corrected by the assumption that one "aggregate" represents, on the average, 2·5 "globular" particles.
PLATE 1. Bovine serum albumin (pH 6·9). (b) Doublet particles.
X
200,000. (a) Typical singlet globular particles.
[facing p. 448
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II. Bovine serum albumin, pH 1·9.
X
200,000.
..
PLATE III. Exam pl es of types of bovine se r u m a lb umin parti cle. (b ) " a ggrega t e" ; (e) "semi-native " ; (d) " t h rea d lik e" .
X
160 ,000. (a) "Globu lar";
PLATE
IV. Bovine serum albumin.
X
160,000. (a) pH 4·2, (b) pH 3·8, (c) pH Hl.
CONFORMA TIONAL CHANGE OF SERUM ALBUMIN TABLE
449
4
Oounts of particle types of B S A at low pH values
%
%
%
%
%
%
pH
Solvent
"Globular"
"Aggregated"
"Semi. native"
"Thread. like"
Native
Denature d
1'9 2·8 3·06 3·12 3·3 3'4 3·6 4·1 4·2
HCl HAc HAc HAc HAc Hel HAc HAc HCl
4·5 20·0 8·0 9·5 2·0 12'5 6·5 34·0 14·5
2·0 17·0 8·0 4:5 4·5 10·5 7·5 19·0 33·5
25·0 1l·0 30·0 18'5 14· 0 30·5 30·0 14·0 38· 5
68·5 52·0 54·0 67·5 79·5 46'5 56·0 33·0 13'5
6·5 37·0 16·0 14·0 6·5 23·0 14·0 53·0 48·0
93·5 63·0 84·0 86·0 93·5 77-0 86·0 47·0 52·0
(Corrected on the assumption that one "Aggregate " represents, on the average, 2·5 "G lobular" particles.)
'" ::>
~
u
(b)
(a)
50
0'" E
'"
.~
0c: 25
'-
0
~
3
5
1 pH
3
5
FIG. 5. Proportions of native molecules ("globular" and "aggregated") in electron micrographs of BSA. Observations corrected on the assumption that one "aggregate" represents, on the average, 2'5 " globular " particles. (a) Preparations from hydrochloric acid solutions. (b) Preparations from acetie acid solutions.
4. Discussion Measurements of particle sizes from shadowed electron micrographs are subject t o unccrtainty in the choice of correction for the accumulation of sha dowing metal; since particle volumes and molecular weights are functions of the cube of linear dimensions, molecular weights obtained from electron microscopy for particles as small as the BSA molecule must be accepted as estimates reliable only to about ± 50%. Attempts to avoid these uncertainties by the use of negative-contrast methods have been unsuccessful (Valentine, 1959). Nevertheless, macromolecules in the 50 to 60 A size range may be studied in shadowed electron micrographs, since it is possible thereby to establish whether particle sizes are in a range consistent with molecular weights as determined by physicochemical methods, and, particularly, to observe directly the over -all shape of particles down to the 20 or 30 A limit imposed by the grain of shadowing metal. 29
450
E. M. SLAYTER
In the present study it has been possible to identify particles of average height 45 A and average diameter 60 A as a major component in electron micrographs of BSA prepared from solutions in the pH range from 4·2 to 6·9. Below pH 4,2, these globular particles are replaced by threadlike particles which, at pH 1,9, have an average diameter of 21 A and average length 250 A. Both of these sets of dimensions are consistent with the established molecular weight of BSA, namely, 66,000. Furthermore, as may be seen from comparison of Figs 4 and 2, the extent of the transition from native to "denatured" BSA observed in electron micrographs, as a function of pH, rather closely parallels the alterations of properties measured in solution. It is encouraging to find that the electron micrographs represent the expected behavior of the molecules as deduced from very extensive physicochemical studies. This result suggests that artifacts of specimen preparation for electron microscopy-including collapse upon dehydration, effects of sudden changes of pH, and disruption by surface tension forces during drying down-need not necessarily preclude the use of micrographs for the interpretation of events as they occur in solution. Nevertheless, certain questions arise concerning the observations described here. It is necessary to explain why anomalous proportions of particle types are observed in preparations made from acetic acid solutions below pH 3·5. The extent to which possible structures of the acid form of albumin can be distinguished from electron micrographs must be considered. The fact that no more than 50% of native particle types are observed in electron micrographs at neutral pH must be accounted for. Finally, the relation of the doublet molecules described by Chatterjee & Chatterjee (1965) to the approximately spherical monomer' found here should be clarified. The apparent re-appearance of native particle types in acetic acid solutions below pH 3·5 may be due to the lowered surface tension of that solvent. As shown in Fig. 6, the effect of HCI upon solvent surface tension is virtually negligible at the highest
(a)
73r7"",-------...,
60~-........--...L.-.~---'
o
4
Weight % acetic acid
72~_--L.._ _':-_....J
o
4
Weight % of Hel
FIG. 6. Surface tension (y in dynes/cm-) of solvent solutions. Arrows indicate maximum weight per cent of each substance employed. Data plotted from Handbook of Ohemistr'lJ and Physics. 45th Ed. 1965, P- F·16. (a) Acetic acid solutions, 30°0. (b) Hydrochloric acid solutions, 20°0,
concentrations used in this study, whereas the highest concentration of acetic acid used was sufficient to lower solvent surface tension by about 7%. The acid form of BSA may no longer be constrained to dry down in extended configuration from solutions of lowered surface tension. The results of Luzzati et al. (1961) indicated a 37% conversion ofBSA from globular
CONFORMATIONAL CH A N G E OF SERUM ALB UMIN
451
to fibrou s form at pH 3·5. It is of interest to consider whether p articles with one or severa l fibrou s regions could be dist inguished from each other, and from native E SA, in electron micrographs. If it were exactly spher ical, t he nati ve E SA molecule would be 54 A in diamet er. If 63 % of the molecule remai ns as a single globular region after a cid-denaturation , t h is port ion would have a molecular weight of 41,500, corresponding to a sphe re 45 A in dia mete r . Since measurements of particle diameters ar e uncertain to about 10 A, the globular portion of the denatured molecule would be essentially indist inguishable from t he original nat ive form. If the fibrous porti on of t he molecule were only partially uncoiled , it might measure about 20 A X 100 A, so that the denatured molecule would appear in micrographs as a " ball and cha in" struct ure. If the fibrous portion of the molecule were a full y extended polypeptide cha in, it would measure less than 10 A in diamet er and might not be det ected. Neither of these possibiliti es is consiste nt with the observat ions. If, alternatively, the acid form of ESA has a structure such as suggested by F oster, in which four globular regions are separated by three fibrous regions, the individual globular regions would have an average molecular weight of 10,500, corresponding to a diameter of 28 A. For incompletely extended fibrous regions, the differen ce in diam et er between "globular " and " fibrous" regions would thus be reduced t o ab out 10 A, and, after coating wit h sha dowing metal, such a molecule would be expected to appear as a more or less uniform t hread. If the fibrou s regions were fully extended polyp eptide chains, the molecule should appear as a series of fairly well-separated spheres ra ther than the threadlike stru ct ure observe d. It is possible, however , that the fibrou s regions might be fully extende d in solution, but par tially contract on dr ying. Such a possibility serves to reconcile the X-ray dat a of Lu zzati et al. (1961) with t he ap pearance of the acid form in micrographs. In summary, while electron micrographs provide no ba sis for suggest ing that there ar e, specifically, four "globul ar " regions in the acid form of ESA, the obser vation of t hreadlike particles in E SA solut ions at low pH indicat es that exte nsion occurs at several points in the molecule ra t her than in a single region. Th e observation, at neutral pH, of occasional doublet st ruct ures and of not more than 50% of native par ticles may be exp lained by t he following hypotheses. ES A exists in solut ion ent irely as t he native configurat ion above pH 4·5 . As shown by titrati on studies (F oster, 1960, p . 212), three hydrogen ions are t aken up per molecule over a narrow range of pH at about pH 4·0. This causes partial loosening of t ertiary str ucture, forming a doublet molecule. The same reaction may rather easily be mimi cked on drying down, by the action of surface t ension or other forces. The doublet molecule is thus considered to represent the expandible form of ESA deduced by Tanford (1961) and Foster (1960). The doublet "subunits" mu st be intermeshed when the molecule is in its fully native form , producing an approximately spherical shape of the latter. The observation of some " t hreadlike " particles in preparations at pH 6·0 indi cates that not only doubl ets but fully extended configurations are form ed from nati ve E SA as an artifact of drying. Table 2 shows t hat, at pH 4,2, 5·8 a nd 6,9, approximately 50 % of molecules ar e observed in micrographs as " nat ive" E SA, 35 % as " semi-native" (including doublets) and 15% as "threadlike". If one accordingly assumes that , at any pH, 50% of native molecules will be converted on dr ying t o the "semi-na t ive" form , and also that 30% of "s emi-native" par ticles, both pre-exist ing and newly formed , will at the sam e tim e be conver ted to the " t hreadlike " form , it is possible to est ima te t he proportions of
452
E. M. SLAYTER
particles present in solution from those observed in electron micrographs. The following equations apply: (1) N; = 2No
Ss =
So -0,7 No --=---,-------=0·7
T s = To -3/7 So
(2) (3)
in which No, So and To are proportions of native "semi-native" and "threadlike" molecules counted in electron micrographs, and N s, Ss and T s are the proportions of the corresponding particle types in solution. Application of this treatment to the results in Table 3 yields Fig. 7, which illustrates the estimated proportion of "doublets" ("semi-native" particles) in solution. Figure 7, in excellent agreement with
'" .2!
50
u
.~ 25 .....o
o
Q.
~
357 pH
FIG. 7. Estimated proportions of doublet ("semi-native") particles in solution as a function of pH.
the predictions of physicochemical studies except for an anomalous value at pH 1,9, illustrates the sudden appearance of a population of "semi-native" molecules at pH 4, and its more gradual decay as the pH is lowered. I thank Miss F. Cotton and Mrs L. Dow for technical assistance, and Mrs M. Federbush and Dr A. D. Dingle for counts of particle types. This work was supported by a grant (GM-09959) from the National Institutes of Health. REFERENCES Chatterjee, A. & Chatterjee, S. N. (1965). J. Mol. Biol. 11, 432. Foster, J. F. (1960). In The Plasma Proteins, vol. 1, ed. by F. W. Putnam, p. 206. New York & London: Academic Press. Hall, C. E. (1956). J. BiophY8. Biochem. Oytol. 2, 625. Hall, C. E. (1960). J. Biophy8. Biochem. Oytol. 7, 613. Harrington, W. F., Johnson, P. & Ottewill, R. H. (1956). Biochem. J. 62, 569. Hartley, R. W., Jr., Peterson, E. A. & Sober, H. A. (1962). Biochemi8try, 1, 60. Luzzati, V., Witz, J. & Nicolaieff, A. (1961). J. Mol. Biol. 3, 379. Reichmann, M. & Charlwood, P. (1954). Oanad. J. Ohem, 32, 1092. Schultze, H. E., Heimburger, N. & Frank, G. (1962). Biochem. Z. 336, 388. Tanford, C. (1961). Phqsical Chemistrs] oj Macromolecules, New York & London: John Wiley & Sons. Valentine, R. C. (1959). Nature, 184, 1838. Yang, J. T. & Foster, J. F. (1954). J. Amer. Ohern, Soc. 76, 1588.