The molecular properties of ApoA-I from human high density lipoprotein.

Abstract The molecular properties of the principal protein component of human high density lipoprotein have been evaluated in aqueous solution at pH 2.0, 7.4, and 12.0 and in guanidine hydrochloride at pH 7.4. The high helical content observed at neutral pH by circular dichroism is partly eliminated in acid, more extensively lost in alkali, and almost completely lost in 1.7 m guanidine hydrochloride. The fluorescence properties of the tryptophanyl residues also indicate greater loss of structure in 1.7 m guanidine hydrochloride than in aqueous solutions at any pH. ApoA-I contains both secondary and tertiary structure in water at neutral pH comparable to that found in native globular proteins and undergoes molecular transitions below pH ∼7, above pH ∼11, and in guanidine hydrochloride solutions between 0.8 to 1.4 m. The properties of succinylated apoA-I at neutral pH resemble those of apoA-I at pH 12.0.

The molecular properties of the principal protein component of human high density lipoprotein have been evaluated in aqueous solution at pH 2.0, 7.4, and 12.0 and in guanidine hydrochloride at pH 7.4. The high helical content observed at neutral pH by circular dichroism is partly eliminated in acid, more extensively lost in alkali, and almost completely lost in 1.7 M guanidine hydrochloride.
The fluorescence properties of the tryptophanyl residues also indicate greater loss of structure in 1.7 M guanidine hydrochloride than in aqueous solutions at any pH.
ApoA-I contains both secondary and tertiary structure in water at neutral pH comparable to that found in native globular proteins and undergoes molecular transitions below pH -7, above pH -11, and in guanidine hydrochloride solutions between 0.8 to 1.4 M. The properties of succinylated apoA-I at neutral pH resemble those of apoA-I at pH 12.0.
The elucidation of the physical chemical properties of the plasma lipoproteins and their apoproteins should facilitate our understanding of lipid metabolism and afford insight into the problems of hyperlipoproteinemia and atherosclerosis. The nature of the interactions between the lipid moieties and apoproteins is of interest in itself and could also shed light on the nature of lipid-protein interactions in membranes. The present study was initiated to compare the properties of the major apoprotein of human HDL' when free of lipid and when combined with lipid in the native state.
In previous work on the properties of the apoproteins, an unfractionated mixture of apoproteins was generally used. It is clearly necessary to characterize the individual apoproteins before one can understand their structure in the native lipoprotein.
Recent studies have shown that apoA-I comprises approximately 65 to 70% by weight of the total protein of HDL (7,13,14). It has a molecular weight of 25,000 to 28,000 (6,12) with NH*-terminal aspartic acid and COOH-terminal glutamine (2, 4). The sequence of the initial 39 residues (15) and the separation of the cyanogen bromide peptides (16) have been reported. In this report we have evaluated some of the molecular and structural properties of highly purified apoA-I. MATERIALS AND METHODS Plasma for the isolation of HDL was collected from a single normal male fC. Z.) bv nlasmanhoresis in EDTA. HDL was prepared by ultracehtri"fuga1 flotation in KBr between densities 1.063 and 1.210 g per ml. The product was delipidated with chloroform-methanol (2:l v/v) as previously described (8). Less than 1% nhosnholinid remained after delipidation. AnoA-I was isolated-from 'the mixture of apoproteins by chromatography of delinidated HDL on Senhadex G-209 in 0.1 M Tris-HCl. 6 M urea. pH 8.5, according to the' method of Scanu et al. (13). The eluates were monitored by disc gel electrophoresis and amino acid analysis. In these studies the leading edge of the apoA-I peak was consistently homogeneous by disc gel electrophoresis, whereas the descending edge was frequently contaminated by apoA-II. The purified apoA-I was dialyzed free of urea and lyophilized.
The degree of purity was evaluated by polyacrylamide disc gel electrophoresis and NHt-terminal amino acid analysis. A single band was seen on electrophoresis and a single residue, aspa& acid, was found for the NHz-terminal residue. Polvacrvlamide eel electrophoresis was performed in 10% gels contaming M urea-at pH 9.4 (17). NH1-terminal residues were determined by the t,hree-stage phenylisothiocyanate procedure of Edman (18) and the phenylthiohydantoins were identified by gas-liquid chromatography (19).
Succinvlation of aDoA-was nerformed in 0.1 M NaHCOn. DH 8.0, at 4' using a 501fold excess of succinic anhydride over'&ee amino groups (20). After 1 hour, apoA-I was resolved from the reaction mixture by gel filtration on Sephadex G-10 in 0.05 M NHnHCOa, and lyophilized.
The extent of modification of the lysine residues was determined by total enzymatic digestion of succinylated apoA-I (8), followed by amino acid analysis.

Structure at Neutral pli
The secondary structure of proteins can be estimated by CD measurements in the far ultraviolet wavelength region (27, 28). The CD spectrum of apoA-I at pH 7.4 (Fig. 2) (in 0.002 M phosphatej is in good agreement with that previously published by Scanu et al. (13) and Lux et ~2. (29). The characteristic minima at 220 and 208 nm and the large negative ellipticities indicate a high helical context. Lux et al. (29) have estimated that about 55% of the peptide groups are in helical segments. The maximum amount, of helix formation was estimated by measuring the CD spectrum of apoA-I in 2-chloroethanol-Hz0 (85:15, v/v), a solvent known to favor strongly helix formation (30) (Fig. 3). corrected to water at 20", was 2.23. This value can be assumed to be the sedimentation constant since the concentration of apoA-I was very low in this experiment.
A frictional coefficient of 1.39 was calculated from this sedimentation constant and a molecular weight of 26,600. with that observed with many native proteins and is considerably blue-shifted with respect to simple tryptophanyl peptides in aqueous solution (32). A value of 0.130 was measured for the polarization at 25".

Conjormational Changes
It is evident that apoA-I contains organized elements similar to those found in most proteins.
The extent of this organized structure was investigated by examining the stability of apoA-I to various conditions and reagents which are known to unfold and denature native proteins.
EJecfs of Acid-The properties of apoA-I in acid were evaluated by the fluorescence behavior of the tryptophanyl residues. When the pH is reduced from 7.4 to 2.3, the wavelength of the emission peak of apoA-I shifts from 333 nm to 338 nm (Fig. 4).
The partial normalization of the emission peak is accompanied by a fall in tryptophanyl polarization from 0.130 to 0.072 between pH 7.4 and 2.2 at 25".
The pH dependence of the acid transition could not be ascertained since unusual time effects indicating a complex reaction were observed. Some of these effects are shown in Fig. 5. The unusual kinetics may result from several overlapping molecular transitions which could affect the fluorescence of each of the four tryptophanyl residues in apoA-I differently.
The situation is even more complicated than indicated in Fig. 5 since data obtained at pH 5.0 and 4.0 (not shown in Fig. 5) had final fluorescence values which were between those observed at pH 3.0 and 2.5. The unusual behavior may be due to the formation of soluble complexes of unfolded forms of apoA-I as the minimum solubility for t.his protein is near pH 4.5 to 5.0 (Fig. 1).
A fall in polarization and red shift in fluorescence usually represent unfolding of the polypeptide chain to a more open, flexible form. This point of view was confirmed by sedimentation velocity and circular dichroic measurements.
The sedimentation rate of a dilute soluticm of apoA-I (11 PM) at pH 2.  1.96 (s~",~~,). The far ultraviolet CI) spectrum of apoA-I at pII 1.7 was similar in shape to that observed at neutral pII, though the negative cllipticbity values at the two minima, 208 and 220, decreased b? almost 500/, (Fig. 2).
Ejfects oj fllh-nli&Vcry little, if any, molecular modification occurs in apoA-I between neutrality and pH -11. Significant changes in structural parameters were observed, however, between pI1 11 and 12, and indicate that apoA-I is more cstensively disorganized at pII 12 than at pII 2.0. The emission peak is shifted from 333 In at pI1 11.0 to 348 nm at pII 12 (Fig. 4). The peak value at 348 nm is close to that found in simple tryptophanyl pcptitles in water (33) (Fig. 4) and indicates that the tryptophanyl residues in apoX-are now highly esposcd to the solvent.
The fluorescence intensity of the tryptophanyl residues, however, is continuously quenched beginning at pH 9. The quenching below pH 11 is aImoFt certainly due to energy transfer to t,he phenolic groups which ionize in this pH region (34). The shift in emission peak, however, results from a change in the environment of the tryptophanyl residues. l'hc pII dependence of tryptophanyl polarization of apoA-I supports the above analysis of the fluorescence data in that there is no ctiaiige until pII -11 and a large decrease by pI1 12 (Fig. 6).
The structural disorganization observed by polarization was confirmed by sedimentatioli and circular dichroism. The sedimentation coefficient of apoA-I declines to a value of 1.82 (sZO,,,) at pH 11.5. The optical activity of the peptide c~hromophore is al>o strongly reduced brtwccn pH 11 and 12. The pI1 depcndence of t,he mean residue cllipticity at 220 nm agree's with that of the polarizat,ion data (Fig. 6). There is evidently a distinct molecular transition above pH 11.

Succinylaled
ApoA-I-Succinylation of the t-amino groups of a protein is used frequently to increase solubility.
The cstent of structural change produced by the increase in negat,ivc charge due to succinylation depends very much on the particular protein investigated (35, 36). Many proteins arc unfolded but a few appear to remain native with extensivr succinylation.
In the case of apoA-I the parameters characterizing the helical structure and the molecular dimensions of the nonsuccinylated molecule have been significantly affected. Judging from the (:I) activity (Fig. 3), polarization, and sedimentation coefficient ( Table I)  the same degree of disorganization as when apoA-I is brought from neutrality to pH 12.

ICflects of Guanidine
Hydrochloride-Solutions of 6 M Gu-HCl convert most globular proteins to random-coil polypeptides (37, 38). The relative stability of a protein can be evaluated from the concentration of Gu-HCl needed for 50% denaturation. The effect of Gu-HCl on the fluorescence intensity and polarization is seen in Fig. 7. It is clear that a molecular transition occurs bctwcen 0.8 and 1.4 M Gu-HCl nit11 a midpoint near 1.1 M.
The normalization of the tryptophanyl emission peak and the small final polarization value indicate estensive loss of structurc. This result was confirmed by the effect of Gu-HCl on the absorpt,ion spectrum of apo.4-I.
A blue-shifted difference absorption spectrum was found in 1.6 M Gu-I-ICI at pH 7.0 with two peaks at 292 and 285 nm (Fig. 8) which are characteristic of tryptophanyl and tyrosyl groups, respectively (39). There was a 7% decrease in absorption at the 292.nm peak, i.e. AA292,/ 1128".
The helical content of apoA-I is also largely eliminated in 1.7 M Gn-HCl.
There are considerable data 011 the properties of the apoproteins of HI)L and on the reconstituted forms of HDL obtained In contrast to the instability of apoA-I below pH 7 there is no evidence of a conformational change between pH 7 and 11. There are, however, important structural changes between pH 11 and 12. 0.000 The molecular parameters indicate that less struc-I I I ture remains at pH 12.0 than at pH 2.0. The smaller values of the polarization, the mean residue ellipticities (at 220 and 208 nm), and sedimentation coefficient indicate greater flexibility of the polypeptide chain at pH 11.5 (Table I).
Relatively low concentrations of Gu-HCl at neutral pH are sufficient to eliminate most of the noncovalent interactions responsible for the globular form of apoA-I.
The polarization and circular dichroic activity in 1.7 M Gu-HCl are reduced to values smaller than those observed in either acid or alkali. The molecular transition in 1.7 M Gu-HCl is also revealed by an enhanced exposure of tyrosyl and tryptophanyl residues to the solvent,, as seen by the denaturation blue shift in the difference -0.020 I I 1 absorption measurements. Absorbance of water solution at 280 nm effect of succinylation is, however, less disruptive on the strucwas 0.270. Pathlength, 1 cm. ture of apoA-I than that of 1.7 M Gu-HCl or the effects of pH greater than 11.5 (Table I).
For the most part these data represent the properties of the mixture of the two major apoproteins (apoA-I and apoA-II) which are isolated directly from the delipidated protein. Scanu (26) has measured the solubility of t,he apoproteins of HDL in solut,ions containing 10 mg per ml. The apoproteins were the least soluble between pH 4.0 and 6.0 where they formed gels and quite soluble above pH 8.0. The solubility of apoA-I is less t,han 0.055 mg per ml (2.2 PM) between 3.8 and 5.8. The loss in solubility may result from the structural disorganization occurring in this pH zone.
The molecular structure of apoA-I at neutral pH resembles that of many native globular proteins in that it has a large percentage of helical residues and a relaxation time large enough to indicate extensive folding of the polypeptide chain. The magnitude of the difference absorption data in 1.7 M Gu-HCl and the rather low wavelength maximum of tryptophanyl emission also reflect significant tertiary structure.
The molecule is ApoA-I is isolated from the other apoproteins found in HDL by chromatography in 8 M urea (13). In this solvent, apoA-I should have very little, if any, organized structure.
In fact, the optical rotatory and viscosity behavior of the apoproteins of HDL are almost the same in 8 M urea as in 8 M Gu-HCl (42). The extensive secondary and tertiary structure that is observed at neutral pH in water indicates that apoA-I refolds when urea is eliminated.
It appears to be quite stable to pH between PH -7 and 11 and to guanidine at concentrations below 1 M. It is less stable to guanidine denaturation than other well characterized proteins, such as ribonuclease or lysozyme (43,44).