Microheterogeneity in albumin: A contaminant

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Microheterogeneity RAPIER Departments

of Biochemistry

and

I!?!$

635-@3

(1967)

in Albumin: H. McMENAMY

Surgery, Memorial

Received

YONG

AND

State University Hospital, Buffalo,

May

A Contaminant’

26, 1967; accepted

of New New June

LEE

York York

at Bufalo,

Edward

J.

Meyer

23, 1967

Bovine Fraction V albumin in 3.3 M KC1 has been subfractionated by lowering the the pH from 4.7 to 3.8. The association constants of skatole with nondefatted subfractions showed differences as large as twofold. Upon defatting, these differences disappeared. It was also found that variations in the pH solubility profiles of the subfractions were evident when albumin was not defatted, or when oleic acid or decanol were added to defatted albumin. Upon defatting the albumin, or omitting the additives (and allowing for variations in dimer content), all differences in solubility of the subfractions disappeared. No evidence was found for intrinsic heterogeneity in the defatted albumin. Details of a modified Goodman defatting procedure are given.

Petersen and Fost,er (12) found that, albumin precipitates from solutions of high salt concentration when t’he pH is lowered from 4.7 to 3.8. It was reported that the fractions precipitating at different pH values within this region differ from each other in t’heir pH solubility profiles and other properties (2, 13, 17). On this basis it has heel1 postulated that albumin is heterogenous; that subtle microdifferences exist in t,he secondary and tertiary structures possibl) through randomization of the amide groups or the disulfide linkages. The suggestion t,hat nat’ive albumin is composedof many different forms with small structural differences between the forms has far-reaching implications. In particular, if these differences are indeed due t,o dist’inct variations in chemical bonds, such as differences among the disulfide linkages, t,hen it, is implied that genetic or intrinsic mechanisms do not have exclusive control over protein t’ertiary structure formation. The basis of this is that such control would not allow multiplicity in the forms of the protein 1 This investigat,ion was supported Public Health Service Research Grant from the National Institute of General Sciences.

by U.S. GM-08361 Medical 635

structures such as the experiments suggest. Rather, if these alterations in chemical bonds are of wide occurrence in albumin, then solution or environmental forces most likely play an important role in the folding of the newly synt’hesized peptide chain. The rationale here is that these latter forces would likely he satisified by a number of configurations provided they were of approximately similar energy levels. The influences of these different factors on the folding t’he peptide chains have been discussedby Klotz (5), u-ho coined the terms “autoplasticism” to designate genet’ic control and “alloplasticism” to designate control by solution forces. We therefore decided to invest,igate the microheterogeneity of albumin through another of its properties, that of indolc binding. Since the site of indole binding is Imow~ to be very sensitive to even minor tertiaq st)ructural changes, it’ was reasoned that microdifferences in the subfractions of albumin, if they exist, should also be manifested in varied ligand associations. It is well known that albumin has a propensity for binding many types of small molecules, both normal metjabolites of the blood as well as any of a large number of foreign compounds wit,h which the protein

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636

may come in contact. The association of small compounds with albumin is also known to alter the properties of the albumin. This is particularly true with lipophilic substances such as fatty acids. Our experimental approach has been to test albumin before and after removal of lipid-like substances, the latter by a modified Goodman defatting process (3). This defatting process differs from that used by Foster and co-workers, who exposed albumin to acid pH conditions to remove lipid-like substances. By following the Goodman extraction procedure we have been unable to detect any differences in indole binding (skatole) among subfractions of albumin. Furthermore, in contrast to what was earlier reported, we have subsequently been unable to detect solubility differences among subfractions of monomeric albumin defatted by the Goodman procedure. By these criteria we therefore believe albumin is not microheterogeneous. Moreover, on the basis of these findings the evidence is entirely consistent that the formation of the tertiary structure of albumin is under exclusive control of intrinsic or autoplastic processes. MATERIALS

Bovine plasma albumin Fraction V (Armour and Co. Lot A21505) was dialyzed with a rapid thin-layer technique against large volumes of 1 mu EDTA, deionized by a mixed-bed resin exchange column, lyophilized, and stored at -20’. This albumin contains approximately 2% dimer by ultracentrifugation and about 2% a-globulin by electrophoresis. Analysis indicates the presence of 0.24 mole free fatty acid per mole albumin. Heptane and pentane (Eastman Organic Chemicals Catalogue Nos. 2215 and P1501, respectively) were distilled before use. Glacial acetic acid (“Baker Analyzed” reagent grade) was fractionally crystallized before use (15). In this method acetic acid was placed in a glass container and agitated slowly at 2” until approximately two-thirds of the solution had crystallized. The liquid was then decanted, the crystals were redissolved by warming, and the crystallization step was repeated. The liquid from the final crystals was used. All other chemicals were at least reagent grade. Prior to use, NatSOl was dried by heating at 200”.

AND LEE METHODS For the deionizing column Amberlite IRA-400 CP-medium porosity (Mallinckrodt), the anionexchange resin, was prepared by placing in a chromatograph column and eluting with 6 N NaOH (10-15 volumesover a 3- to 5-day period) until a negative test for Cl- was obtained. The resin was then washed with distilled water until neutral and stored at 4” in water until used. Amberlite IR-120 CP-medium porosity (Mallinckrodt), the cationexchange resin for the deionizing column, was extracted in a Soxhlet extractor with 6 N KC1 for approximately 10 hours (until no further shedding of color in fresh HCl solution was observed). It was then washed with water until free of Cl- and stored in water at 4’. In preparing the column, a one-half inch layer of cation resin was placed at the bottom of a Corning type (No. 38450) chromatographic column. A wet resin mixture of two parts anion and one part cation resin was added next. The mixed resin height was at least 5 inches and was always added in at least tenfold excess over that calculated for the removal of the salt present. Subfractionation of albumin was conducted by the method of Petersen and Foster (12). Isoionic albumin was dissolved in 3.3 M KC1 and its concentration was adjusted to 0.280.32 gm albumin/ 106 ml on the basis of E:zm = 0.667 at 280 mp. (Where comparisons were made, concentrations of the paired solutions were adjusted to within 0.0015Q/o of each other.) Hydrochloric acid, 0.1 M in 3.3 M KCl, was added for fractionation at 18”. Fifteen minutes after HCl addition the pH was recorded by a Radiometer pH meter 26. The amount of protein in the solution was determined by absorption spectroscopy on an aliquot of the supernatant solution obtained either by suction through a sealing tube with a fine grade filter disc as earlier described (12), or by centrifugation where a clear supernatant solution was obtained. The fractions desired were collected by centrifugation. Usually two fractions were saved: roughly the first and last third to precipitate. Fractionation was usually completed within 3-4 hours. At the end of this period the precipitates were immediately dissolved in 0.05 M EDTA and the pH was adjusted to approximately 6. At this point the solutions were allowed to stand overnight. The next day they were passed through Sephadex G-25 (to remove gross amounts of salts), followed by a small deionizing column, and lyophilized. For restudy the fractions were dissolved in 3.3 M KCl, and HCl was added as described above. Defatted albumin was prepared by a modified method of Goodman (3). The apparatus is shown

MICROHETEROGENEITY

IN ALBUMIN:

FIG. 1. They heptane-acetic acid defatting column. The chromatographic column is Corning type 33456, 400 X 20 mm; the reservoir is a liter round-bottom flask; the stoppers are neoprene; and all tubing is Tygon tubing except end A, which is heavy wall rubber tubing. Depending on the

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637

in Fig. 1. Protein powder was placed in the chromatographic column, a vacuum pump with a solid COz-acetone trap was connected to outlet A (clamp B is closed, clamps C and D are open), and the system was evacuated for a minimum of 6 hours. Clamp A was then closed to maintain vacuum, and the apparatus was transported to a 4” cold room and allowed to stand 16-15 minutes. (A -20” cold room was used in some studies.) At the latter temperature, leaks frequently occurred in the defatting apparatus due to shrinkage of stoppers. Experiments at this temperature were conducted only to the extent necessary to demonstrate that the final product was not different from that obtained at 4”). With clamp C closed a mixture of acetic acid and heptane (1:20, v/v) dried over NazSOI was allowed to enter the evacuated column by suction through line B to attain a height of 4 inches or twice that of the protein powder in the column. Clamp B was closed, and clamp C was then adjusted to allow the acetic acidheptane mixture to flow into reservoir D at a rate of l-2 ml/minute. When the heptane-acetic acid solution reached the bottom of the column, the column was refilled to the previous level and allowed to stand overnight. The next day the column was emptied, refilled, and extracted once more with heptane-acetic acid solution. Four extractions with dried heptane were then carried out. Care should be taken that the solvents do not come in contact with the rubber stoppers. With a leakfree system and careful manipulation of clamp B to avoid loss of vacuum in filling, there was no difficulty in extracting with the solvents until the reservoir was two-thirds full. On the other hand, if difficulty is encountered in slow flow rates, clamps C and D may be closed and the reservoir may be detached between clamp C and D, emptied, evacuated, and reattached. After the final alkane extraction, clamp C was closed, the reservoir was removed, the column was rapidly connected to a vacuum pump with a solid CO*-acetone trap, and a vacuum was applied 5-6 hours. Following this the protein powder was dissolved in water at 4” in the presence of 5 ml mixed resin per gram of albumin. The solution was then passed immediately through a mixed-bed resin column (10 ml wet resin per gram protein) and freeze-dried. The freeze-drying step is very important in removing 5-10 molecules/mole albumin tenaciously held heptane. When this procedure is followed, there is bulk volume of the protein, which should not be greater than half the volume of the column, other sizes of chromatographic columns can be used. In such cases flow rates should be appropriately readjusted.

638

McMENAMY

AND

LEE RESULTS

Lo! 4.4 PH

FIG. 2. The pH solubi1it.y profiles of various subfractions of bovine albumin. A, Uufractionatcd nondefatted; 0, nondefatted F (4-22%); l , nondefatted F(70-96%) ; X, unfract.ionat.ed defatted albumin. Dimer content 226%.

little increase in dimer content and the product is readily soluble in water. Analyses have shown that the albumin so prepared contains none of the added acetic acid (3) and less than 1 mole alkane/ mole albumin (11). Pentane has also been used in lieu of heptane in the extract,ion procedure wit,h no difference observed in the final product. Fatt,y acid analyses were kindly determined for us by Dr. David Schnatz according to his own method (14). Dimer content was determined with a Sephadex GlOO colmnn (180 X 10 mm) using essentially the technique reported by Whitaker (18). The degree of dimer content, was estimated from areas under the 280 1~11 tracing from a Gilson fract,ionator and a Texas Instjrument recorder. Bin&g silLdies with albumin were conducted by the method of centrifugal ultrafiltration (8, 9). One- to Z-ml albiimin solritions (0.12 f 0.01 mM) containing 1.5, 1, 0.67, 0.5, 0.35, and 0 moles skatale/mole albumin in 0.05 M EDTA and 0.1 M KCl, pH 63, were placed inside semipermable membranes which were then suspended in tubes and centrifuged to obtain ultrafiltrates. The moles skatole bound per mole albumin (0) was computed for each concentration studied (0 mole skatole was used as a blank). In a plot of s/A versus 0 (where A is the concentration of skatole) the intercept and on the D axis is in, Since on the B axis is Zk,n,‘, lzi is 1 the association constant is given directly by the S//I intercept,.

pH Precipitation prq/iles. The subfractions of albumin are designated according to the order of t,heir precipitation on the addition of HCl. I’or example, I~(&22 %) means t,hat the prot,ein in t,he fraction is 18 % of the total protein in t)he experiment, and that it, is the 4-22 % part, in order of precipitation on addition of HCl. It, is clear in l’ig. 2 t,hat 0.1-0.2 pH unit separate t,he soluhilit!. profiles of the t\vo subfractions, 1~(+22 %) and 1:(70-96 %), ohtained from nondef:rtted albumin. On the other hand, after defatting (Fig. 3) the soluhility profiles of the same t,\\-o sukfrtlctions become indistinguishable from each other. They are :kw indist~inguishablc from the solubilit,y profile of unfraction:~t,ed dcfatted albumin. A comparison of Figs. 2 und :3 shows that dcfat,ting has not altered the profile of lc(4p22%), which contained no fatt.v acid at the start, (Table I). The dimer content, of the three proteins studied in Fig. 3 was ?A %. Ht~ck tit,rations of defnkd I:(& 22%) and defatted P’(70-96%), in \vhich 0.1 M KOH in 3.3 M KC1 \vns used, are also she\\-n. l+hcn though 5 16 hours with slow agitxtion \vas :Lllo\ved aft’er e:rch pH adjust-

-5,--1 5.8

PH

FIG. 3. The pH solubility profiles of various subfractions of bovine albumin. 0, Defatted F (4-22%) ; A, def atted F (7@96%). Back titrations with KOH of defatted F(4-22%) and defatted F(70-96%) are shown by the symbols l and A, respectively. Dimer content 3-570.

MICROHETEROGENEITY

IN

ment, the back-titration profiles nevertheless showed a large hysteresis effect. Complete solubility was not obtained until pH 5.67 was reached. Figure 4 shows experiments where the albumin used in fractionation was defatt#ed albumin. In study A there was no difference in the solubility profiles of the first and third subfractions. Here t,he first fracGon, F(252 %), contained half of the original albumin, and its dimer content was not much different from the last fraction, I’(79-99%). In a second experiment, study B, t’here was a difference of about 0.02 pH unit bet,ween t#he two subfractions, F(2-20 %) and F(51-98 %). Because of the positions at which the latt#er subfractions were removed the difference in t)heir dimer content was 8 %. This experiment suggested that differences in the solubility profiles of the subfractions may result from differences in the dimer content. I’et,ersen and I>oster (12) had reported a study xv-here 45% dimerized albumin precipit,ated at a pH approximately 0.08 unit higher t,han 7 % dimerized albumin. To further evaluate the dimer effect, a solubility study was conducted with defatted crystalline albumin Fvhich contained approximately 30 YG dimer and 10 % higher order aggregat,es (Fig. 5). There was approximat,elv 0.1 pH unit, differences between the solubiiity profile of t,his albumin and that of TABLE F.~TTY

Fraction Fraction F(4-22%) F(70-96%)

I

ACID CONTENT OF BOVINE ALBUMIN FRMTIONS

V V defatted nondefatted nondefatted

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: A CONTAMINANT

4.0

4.2

4.4

4.0

4.2

4.4

PH

FIG. 4. The pH solubility profiles of bovine albumin defatted before fractionation. Study A: X, unfractionated albumin; 0, F(2-52%), 0, F(79-99%); dimer content 4, 5, and 3y0,, respectively. Study B: 0, F(2-20%); 0, F(51-98%); dimer content 11 and 370, respectively.

A -z E \ h

0.2-

% .< 2 5 2 e

0.1 -

0.24 -0 -0 0.9

a Defatted preparations assayed by the isopropanol-heptane-H2304 extraction procedure (14) consistently gave readings for free fatty acids in the range of 0.15-0.23 mole fatty acids/mole albumin. Reextraction of the albumin did not further decrease this value. For this reason it has been considered that a value of -0.2 represents an “albumin blank,” induced by sources other than free fatty acid. In view of this the values as reported in this table have been reduced by 0.2 unit.

PH

FIG. 5. The effect of dimer content on the solubility profile. Study A: 0, defatted crystalline bovine albumin (lot 10 Pentex), contains 40% dimer and 10% higher order aggregate. The dashed line is the profile for defatted Cohn Fraction V albumin, which contains 4% dimer. Study B: 0, defatted F(2-5201,) with the dimer content increased to 12%; 0, defatted F(79-99%) with the dimer content 3%.

640

McMENAMY

PH

the 0, 0, X,

FIG, 6. The effect of oleic acid and decanol on pH solubility profile of defatted albumin: 0.33 mole oleic acid added per mole albumin; 0.33 mole decanol added per mole albumin; defatted albumin no additives. Dimer content,

3-5%. defatted albumin containing 4% dimer. In another study the albumin fraction F(252 %) was allowed to partially dimerize by exposure t,o acid PH. There was approximately 0.02 pH unit between the solubility profile of this fraction which contained 12% dimer and F(79-99 %), where the dimer cont,entf was 3%. It is apparent by these comparisons that the difference in dimer content between the two subfractions in Fig. 4B is approximately of the correct order to account for t,he difference in the solubility profiles of these two subfractions. Figure 6 shows pH titration profiles of albumin to which oleic acid and decanol were added. Even at the low levels of the substances added, the pH profiles are distinctly altered, especially at the lower part of the profile. However, to the extent of the fatty acid present, the solubility profile of the preparation with added fatty acid is not altered as much as that of the original nondefatted albumin (compare Figs. 2 and 6). &zding studies. Two gm of bovine Fraction V albumin (nondefatted) was frac-

AND LEE

tionated into four parts: F(O-9%), F(922 %), F(22-67 %), and F(67-96 %). These fractions were dissolved in EDTA buffer, pH 6.7, desalted by passagethrough a Sephadex G-25 column followed by a mixed-bed resin column, and finally lyophilized. The binding of t’hese fractions with skatole was studied (Fig. 7). It was found that F(67-96 %) bound with a considerable lower affinity than the other fractions. Upon defatting, however, this fraction then bound as well as the other fractions. Fraction F(O9 %) was also defatted. The association with this fraction (which presumably contained no fatty acid) was unchanged by the defatting process. Other observations.Dialysis of nondefatted F(4-22 %) against nondefatted F(70-96 %) in 0.1 M acetate buffer (pH 4.2) for 24 hours, using a rapid thin-layer technique, did not change the solubility profiles of the two subfractions. A similar experiment conducted with 4 M urea present causeda large broadening of the pH solubility profile of F(4-22%) and a small broadening of F(70-96 %). When F(70-96%) was defatted and placed in 4 M

uFIG. 7. The binding of skatole with different subfractions of albumin (pH 6.3), 0.1 M NaCl, 0.05 EDTA 4”; 0, unfractionated nondefatted albumin; & nondefatted F(O-9%); A, nondefatted F(9-22%); X, nondefatted F(22-67%); 0, nondefatted F(67-96%); 0, defatted F(O-9%); a, defatted F(67-96%). The binding constant is given by the O/A intercept. The molecular weight of albumin was taken as 64,300.

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IN ALBUMIN:

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Gil

initially defatted were nonexistent or very small. The first fraction separated on acidification contained more dimer than the later fractions. The differences sometimes observed between the solubility profiles of the subfractions of defatted albumin were of the order one expects from the differences in dimer content. The first fraction separated also contained some tightly bound dark mat’erial (presumably heme or heme products), a suggestion t’hat this contaminant might also alter the solubility profiles-although it is obvious that its contribution was not very large. In view of all this it seemsmost likely that the microheterogeneity sometimes noted in monomeric albumin is due to contaminants (in a large part fatty acids) which can be ext’racted by a proper defat,ting process. It is well known that albumin contains a nonstoichiometric sulfhydryl content; typically there are 0.7 mole SH per mole of albumin. The SH content was not measuredin our present studies: however, it is apparent that its effect on the solubility profile of the monomer, assumingt’hat the 0.7 ratio holds, must be rather small. It is found that while t’here is considerable hysteretic effect in dissolving the prepicitate of the F form of albumin, this form does indeed dissolve with readjustment of pH and reduction of salt content. Interconversion between t,he F form and the N form is complete and wholly reversible. Differences between defatted and nondefatted albumin in the binding of indole compounds was earlier found to be very marked (6, 11). With the defatted albumin, the association constant was considerably higher than with nondefatted albumin. In the pH region 8-10, het’erogeneity in the primary site was alsofound with nondefatted human albumin. This n-as evident from the DISCUSSION fact that part of the albumin in this pH The subfractions separated by the addi- region had a very high association constant, tion of HCl to KC1 solutions of nondefatted whereas the remainder had a low association Fract,ion V albumin demonstrated clear dif- constant. This latter type heterogeneity was ferences in their solubility profiles. These not found in the pH region 5-7 nor, was it differences, however, totally disappeared found with defatted albumin. Small amounts when t,he subfractions were defatted. Solu- of fatty acids t’herefore induce recognizable bilit?; differences among subfractions ob- heterogeneity in albumin at positions other tained from albumins which had been t,han at the N-F transition pH. urea overnight, at pH 4.2, it too showed ext,ensive broadening of the solubility profile. After exposure to 4 M urea both F(4-22%) and defatted F(70-96 %) contained over 60% dimer or multiple aggregate, whereas nondefatted 1;(70-96 %) similarly exposed to 4 M urea contained about’ 20% dimer. These experiments demonstrate that it is the fatty acid (or other bound substances)which provide stability for the albumin. It is also evident, from the dialysis experiment#sthat t’he bound substanceson the protein are st)rongly attached to the protein. It n-as observed in several fract’ionation experiments that, the solubilit’y of albumin n-assomewhat,greater at, 4” than at’ IS”. This, interestingly, is consistent wit#h Ferry’s observat,ion with “sulfated albumin” (albumin made acid to pH 4 with HZSOJ, as reported by I\MIeeken (1939). The latter albumin ~~-assoluble at, O”, but precipitated at 25”. The fluorescence of several of t,he albumin preparat,ions was measured (excit’ation 2SO nq.~,emission365 mp) with the transmission of defatted albumin set as 100%. At the same protein concentration, transmissions of 91, 94, S.5,9S, and 100% were obtained for nondefat,tcd Fraction V, nondefatted F(2-20%), nondefatted F(51-98%), defatted B’(220 %), and defatted F(51-9S %), respectlively. The reduction in fluorescence therefore was approximately proportional t)o the fatty acid cont,ent present. Subfractions of nondefatted albumin did not show an enrichment of dimer in t’he first fraction to as great an extent as that found with defatted albumin. Neither did t’he concentrat,ion of the “dark albumin” earlier not,ed by Hughes and Dintzis (4) and I’etersenand Foster (13) seemto be as great in the first fraction of nondefatted albumin as nit,h defat,ted albumin.

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The defatting procedure discussed above differs little from that used in this laboratory for the last several years. Goodman (3) has shown that acetic acid added in the defatting procedure is removed from the final product, and it has been shown in our laboratory (11) that any remaining alkane is at a very low level, less than 1 mole/mole albumin.2 It is most important that rigorous anhydrous conditions are maintained in defatting or the final product will aggregate and is difficult to dissolve. Nondefatted Fraction V bovine albumin contains approximately 2 % dimer, which typically increases to 3-5% after defatting. We have not been able to decrease this dimer content further. Undoubtedly this is due to the instabi1it.y of defatted albumin. In solution the latter t’ends to aggregat’e much more readily than nondefatted albumin. This instability in defatted albumin is not unexpected since it, is well known that albumin is much less stable in t’he absence of fatty acids than in their presence. The addition of fatty acid to defatted albumin did not alter the pH solubility profile to the extent that a similar amount of “native” fatty acid alters the profile of nondefatted albumin. In addition to there being differences in the t,ypes of fatty acids (we added oleic acid whereas many types of fatty acids are known to be present in the native albumin), there are undoubtedly present on nondefatted albumin, substances other than fatt)y acids, such as cholesterol, cholesterol esters, and triglycerides. These would be expect,ed to modify the solubility profile of the protein and also would be expected to be extracted by the defatting procedure. One would not expect the binding of skatole to be altered by the dimer content in 2 The tritium labeled pentane or n-octane used to demonstrate completeness of removal of alkane from defatted albumin contained a persistent contaminant which was not completely volatilized from surfaces of containers. Through the use of variable levels of activity and solutions without albumin as blanks, it was demonstrated that O-O.7 mole n-octane per mole albumin remain with the albumin. The latter value might well have been zero, but the contaminant made it difficult to evaluate the results more closely.

AND

LEE

the subfraction. It was earlier shown that the binding of indole compounds with albumin was essent,ially insensitive to dimer format’ion (11). A new method for defatting albumin by charcoal absorption has been recent.ly reported by Chen (1). His method undoubt,edly considerably improves t,he purity of the albumin. He has shown that, added fatty acids are removed and that fluorescence is maximum and constant aft’er charcoal treatment in the same manner as we observed with albumin defatted by the Goodman method. However, Sogami and Foster (l(i), in evaluating solubilitjy profiles of the subfractions from albumin purified by charcoal absorption, conclude that, while the albumin so prepared is more homogenous than albumin without charcoal treatment, distinct het’erogeneity (as evidenced by differences in the solubility profiles) nevertheless st’ill remains. On the basis of t,his observation it is suggested that charcoal absorption does not give as homogenous an albumin as that obtained by the Goodman defatting procedure. REFERENCES 1. CHEN, R. F., J. Biol. Chem. 342, 173 (1967). 2. FOSTER, J. F., SOGAMI, M., PETERSEN, H. A., AND LEONARD, W. J., JR., J. Biol. Chem. 240, 2495 (1965). 3. GOODMAN, D. S., J. Am. Chem. Sot. 80, 3892 (1958). 4. HUGHES, W. L., AND DINTZIS, H. M., J. Biol. Chem. 239, 845 (1964). 5. KLOTZ, I. M., Arch. Biochem. Biophys. 116, 92 (1966). 6. KRASNER, J., AND MCMENAMY, R. H., J. Bid. Chem. 241, 4186 (1966). 7. MCMEEKIN, T. L., J. Am. Chem. Sot. 61, 2884 (1939). 8. MCMENAMY, R. H., LUND, C. C., VAN MARCKE, J., AND ONCLEY, J. L., Arch. Biochem. Biophys. 93, 135 (1961). 9. MCMENAMY, R. H., AND SEDER, R. H., J. Biol. Chem. 238, 3241 (1963). 10. MCMENAMY, R. H., J. Biol. Chem. 239, 2835 (1964). 11. MCMENAMY, R. H., J. Biol. Chem. 240, 4235 (1965). 12. PETERSEN, H. A., AND FOSTER, J. F., J. Biol. Chem. 240, 2503 (1965).

MICROHETEROGENEITY 13. PETERSEN, H. A., AND FOSTER, Chem. 240, 3858 (1965). 14. SCHNATZ,

D. J., J. Lipid.

15. SHAPIRO,

J., Anal.

Res.

IN ALBUMIN:

J. F., J. Biol. 6, 483

(1964).

Chem. 39, 280 (1967).

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16. SOGAMI, M., AND FOSTER, J. F., Federation Proc. Abstr. 26, 826 (1967). 17. SOGAMI, M., AND FOSTER, J. F., J. Biol. Chem. 238. PC 2245 (1963). J. R., Ann. Chem. 36, 1950 (1963). 18. WHITAHER,