R.11.ROSENRERG, \v. F. LEVER.AND M. E.LYONS
6.502
THE CNIVERSITY LABORATORY OF PHYSICAL CHEMISTRY RELATED TO H A R V A R D L-YIVERSITY, A N D THE DEPARTMEXT O F DERM.4TOLOGY, HARV.4RD
[COSTRIBUTION FROM
Vol. 77
MEDICINE A N D PUBLICHEALTH, MEDICALSCHOOL]
The Interaction of Human Serum P-Lipoprotein with Certain Small Molecules1~2
w. F. LEVERA4ND11.E. L Y O N S
BY ROBERT AI. ROSENBERC,3
RECEIVEDJUSE 11, 1955 The binding of the anionic dye methyl orange by human serum P-lipoprotein has been studied by spectrophotometric and equilibrium dialysis methods. I t has been found that, a t PH 7.4, P-lipoprotein binds methyl orange more strongly than does bovine serum albumin but less strongly t h a n human serum albumin. This is the only reported erample of a protein which binds anions on the alkaline side of the isoelectric point as strongly as the albumins. Spectrophotometric evidence is presented for the binding of the uncharged dye chrysoidin by 0-lipoprotein.
Introduction There has been much discussion in the recent literature concerning the configurational requirements for the binding of anions by protein^.^-^ One way of testing the hypotheses set forth is to examine the binding properties of a wide variety of pure proteins and try to ccrrelate the relative strength with which a protein binds anions with other properties or characteristics of the protein. Klotz4 has studied the binding of methyl orange ( I ) by a number of purc proteins a n d correlated
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/
11
Experimental
Materials.-The human serum @-lipoprotein was prepared from Fraction 111-0 of human plasma by the method of Oncley, Gurd and Melix~.~ Fraction 111-0 was provided by the laboratories of the Massachusetts Department of Public Health as a by-product in the preparation of albumin13 and y-globulin" from blood collected by the American Red Cross. Phosphate buffer was prepared from reagent grade salts. (C H ~ ) ~ S - ~ - S ~ ~ S - ~ - S O , i The crystalline sodium salt of methyl orange was a product of Merck and Company. Crystalline chrysoidin hydro1 chloride was obtained from National lniline Company. the binding strength with the binding index, a Methods.-The interaction was studied by the method of function of the amino acid composition, based on equilibrium dialysis. X bag of \'isking sausage casing containing 5 ml. of approximately 0.5% protein solution in the concept of competition of the anion with other 0.1 X phosphate buffer a t PH 7.4 was placed in a glass vial groups on the protein for binding sites. Mihalyi8 containing 5 ml. of dye solution in buffer. The vial was has studied the binding of methyl orange by then stoppered with a polyethylene cap and allowed to rotate myosin and found that the results support the in a constant temperature bath a t 0.0 f 0.1' for 16 hours. The time required to reach dialysis equilibrium was deterbinding index hypothesis. mined by rotating for varying lengths of time a vial in which In the course of other studies on human serum a solution of methyl orange and a dialysis bag containing ,&lipoprotein it seemed worthwhile to investigate only buffer were placed. Sixteen hours were found to be the binding of methyl orange by this protein in adequate. Such a vial was also carried through with each of dialysis experiments. The equilibrium concenorder to provide data to test further the hypotheses group tration of methyl orange in the outside solution was deterwhich have been proposed, mined by measurement of the absorbancy of the solution a t Since it has been suggested that P-lipoprotein is a 465 mp in a I-cm. cuvette with a Beckman D U spectrophotometer. The total concentration of dye inside the bag transport medium for charged and uncharged was determined by measuring the absorbancy of the solulipids in the blood ~ t r e a m and , ~ since the binding tion a t the isosbestic point of the spectra of free and bound of neutral molecules has been attributed to the dye and correcting for the absorbancy of the protein a t this same sites on the albumin molecule as the binding wave-length. The free dye concentration inside the bag was of anions,'O spectral studies were also carried out assumed to be the same as the free dye concentration outside the bag, since the Donnan effect should be negligible on the binding of the uncharged dye chrysoidin in solutions of 0.1 d l phosphate for the protein concentration (2,4-diaminoazobenzene) (11) by @-lipoprotein." used. The protein concentration was calculated using a known dilution from a stock solution. The concentration ( 1 ) Supported by a research grant from the United States Public of the stock solution was determined by drying a n aliquot Health Service (RG-3091). t o constant weight in LlaczLo a t 70°, with correction for the (2) Presented before t h e Division of Biological Chemistry at t h e weight of salts in the solution. -1hsorptinn spectra were 124th meeting of t h e American Chemical Society. Chicago, Sepdetermined with a Beckman D U spectrophotnmeter. PH tember, 1953. measurements were made with a Beckman Afodel G pH (3) Hall Laboratory of Chemistry, Wesleyan University, 1IiddIemeter. town, Connecticut. ~
(4) I. M. Klotz and J. XI. Urquhnrt, THISJOURNAL,71, 1397 (1949). ( 5 ) J. 11,Luck, Discs. F a r a d a y SOC.,6, 44 (1949). (6) F. Karush, THISJOURNAL, 72, 2703 (1950). (7) J . Schellman, J . P h y s . Chem., 5 7 , 472 (1953). (8) E. Mihalyi and B. K. Ghosh, Aicii. Biochein. Biophys., 41, 104 (1952). (9) J. L. Oncley, F. R. S. Gurd a n d RI. Xelin, THISJOWRNAI., 72, 458 (1950). (IO) I. h l . Klotz and J . Ayers, ibid.,74, 6178 (1952). (11) With t h e possibility in mind t h a t calcium binding by lipoproteins might account f o r a substantial part of t h e serum calcium which is bound by globulin,lz exploratory experiments were carried out on t h e interaction of calcium with &lipoprotein. a-lipoprotein, fraction 1\*-4la and -,-globulin. I t was found t h a t the lipoproteins and fraction I V - 1 bind approximately t h e same amount of calcium, while t h e binding b y
Results In the presence of O.5Y0 human serum @-lipoprotein the peak in the absorption spectrum of methyl orange was shifted from 465 to 420 m l , y-globulin is substantially smaller. T h e amounts bound were such as t o indicate t h a t none of t h e globulins tested accounts for a disprw portionate amount of t h e serum bound calcium. (12) F. C. 3lcLean and A . B. Hastings, 1.Bioi. C h e m . , 108, 2 8 5 (1933). (13) E. J . Cohn, L. E. Strong, W.L. Hughes, J r . , D. J. RZnlford, J. N. Ashworth, hI. hIelin and H. I.. Taylor, THISJ O C R N A I . , 6 8 , 459 (1946). (14) J . L Oncley, X I . RIelin, D. 4 . Richert, J TI'. Cameron and P. M. Gross, J r . , i b i 4 . 71, 3 4 1 11949).
Dec. 20, 19~5~5
INTERACTION O F
HUMAN SERUM
@-I,IPOPROTEIN WITH SMALL h~OI,ECUI,ES
6503
as indicated in Fig. 1. This shift is in the same direction as found by Klotz for bovine albumin15 and @-lactoglobulin. When equilibrium dialysis experiments were carried out with p-lipoprotein, the results shown in Fig. 2 were obtained. For purposes of comparison the results are plotted as number of moles of bound dye per lo5 grams of protein, and some data of Klotz and co-workers for binding of methyl orange by bovine serum albumin,I6 human serum albumin4 and p-lactoglobulinJ have been plotted on the same graph. It may be seen that @-lipoprotein binds methyl orange much more strongly than 6-lactoglobulin does and is comparable in binding ability to bovine and human albumin. The experiments with plipoprotein were carried out in higher concentrations of phosphate buffer than those with bovine and human albumin and P-lactoglobulin. Any errors in the comparison would therefore be in the direction of minimizing the binding ability of the lipoprotein. I n the case of the P-lipoprotein, only about 23y0 of the weight of the lipoprotein is due to ~ e p t i d e and , ~ if the results of Fig. 2 were expressed in terms of peptide (or amino acid) content, the binding by @-lipoprotein would be stronger than any of the other proteins mentioned here. 0.2001 I The results of spectrophotometric studies with 400 425 450 475 ROO chrysoidin are shown in Figs. 3 and 4. The change It‘ave length (mp). in the absorption spectrum of the dye in the presence of p-lipoprotein is presumptive evidence for the Fig. 1.-Spectrum of methyl orange i n the presence and abinteraction of this uncharged dye with the protein. sence of &lipoprotein; 0.1 ,If phosphate buffer, PH 7 4. For reasons which will become clear below, the spectra of chrysoidin in the presence of serum albumin, in strongly acid solution, in petroleum ether and in dodecyl alcohol are also shown. 0
HUMAN P-LIPOPROTEIN O’C,pH 7.4
Discussion Up to this time no other globular protein has been found to bind anions on the alkaline side of the isoelectric point as strongly as do the serum albumins. The explanations advanced for the heretofore unique position of the serum albumins in anion binding have been primarily of two kinds. Klotz4 has correlated the binding ability of various proteins with the amino acid composition, while Karush17 has invoked the “configurational adaptability” of serum albumin to account for its affinity for a wide variety of anions. Since no amino acid analyses are available for serum @-lipoprotein,i t is not yet possible to use the data reported here as a test for the “binding index” of Klotz. Furthermore, the presence of 73% by weight of lipid in the lipoprotein molecule9 may make inapplicable a test based on the analysis of the 25% of the molecule composed of peptide chains. I n discussing the “configurational adaptability” of serum albumin KarushI7 correlated the binding ability of albumin for anions with its solubility. @-Lipoprotein is a euglobulin, ivsoluble in the absence of salts a t the isoelectric point,g but still binds anions as strongly as does albumin. This (15) I. M. Klotz, THIS J O U R N A L , 68, 2299 (1946). (16) I. M. Klotz, H. Triwush and F. M. Walker, i b i d . , TO, 2935 (1948). (17) F. Karush. ibid.. 74,2705 (1950).
I 6.0 Fig. 2.-Binding
I
I
I
5.0 4.0 -log (free dye). of methyl orange by P-lipoprotein; 0.1 d l phosphate buffer, pH 7.4.
would seem to throw some doubt on the correlation between solubility and anion binding. I n order to obtain additional information about the binding sites on the lipoprotein molecule, the results were calculated in terms of i , the average number of dye ions bound per mole of lipoprotein, a t each concentration, c, of free dye. The molecular weight of the lipoprotein was taken to be 1,300,000.18 If there are n binding sites with (18) J. L. Oncley, G. Scatchard and A. Brown. Chem., 617 184 (1947).
J. Phys. Colloid
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