Separation of lipoproteins, albumin and γ-globulin by single-step ultracentrifugation of human serum. Application I: Binding of hematoporphyrin to human serum and to albumin

hr. J. Biochem. Cell Bid. Vol. 21, No. 4, pp. 371-384,

0020-711X(94)000883

Separation of by Single-step Application I: Serum and to

1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1357-2725/95 $9.50 + 0.00

Lipoproteins, Albumin and y-Globulin Ultracentrifugation of Human Serum. Binding of Hematoporphyrin to Human Albumin

MAGNE KONGSHAUG,” JOHAN MOAN Department of Biophysics, Institute for Cancer Research, The Norwegian radium Hospital, 0310 Oslo 3, Norway Previous studies of the serum binding of the photosensitizer hematoporphyrin (HP) have given widely different results. The serum binding of Hp is therefore further ilbnninated by experiment and discussion.Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classesand the two major high density proteins (HDP), albumin and y-globulin, were separated in a NaCI-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp-doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (HP/M) was much lower than earlier published ones (106/M) found by other techniques. The association of Hp witb HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreasedwith increasing Hp concentration. The serum binding of Hp is tbe same as that found previously using another rotor and another salt gradient (70.1 Ti, 7O,OOOrpm,NaCf-CsCl). LDL has high-affhtity-lowcapacity binding sites for Hp. HSA is the major HDP protein that bii Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presenceof other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp. Keywords: Ultracentrifugation Serum protein interactions

Serum protein separation Protein-bound hematoporphyrin

Int. J. Biochem. Cell Biol. (1995) 27, 371-384

INTRODUCTION

The use of tetrapyrrole photosensitizers in preclinical and clinical photochemotherapy (PCT) of turnours (Moan and Berg, 1992; Peng et al., 1994; Dougherty and Marcus, 1992) have spurred considerable interest in the binding of such drugs to human plasma or serum (Bare1 et al., 1986; Kessel et al., 1987; Zhou et al., 1988; *To whom all correspondence should be addressed. Received 13 June 1994; accepted 15 November 1994. 371

Mazibe et al., 1991; Kessel and Woodburn, 1993; Kongshaug 1992, 1993; Jori and Reddy, 1993). The serum proteins include the major lipoprotein classes (very low density lipoproteins, VLDL; low density lipoproteins, LDL; high density lipoproteins, HDL) and high density proteins (HDP), the major HDP entities in plasma being albumin and y-globulin. Many tetrapyrroles bind both to lipoproteins and to nonlipoproteins (Kessel et al., 1987; Kongshaug, 1992; Kongshaug et al., 1993). Separation methods that allow simultaneous

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determination of drug binding (in aqueous mileu) to both lipoproteins and nonlipoproteins are therefore required. The use of dialysis is complicated or precluded by adsorption of many tetra-pyrroles to dialysis bags (cf. Kongshaug, 1992). In gel elution chromatography of drug-doped serum, drug adsorption to the gel may occur (Kongshaug, 1992, 1993). Moreover, VLDL and/or LDL are not well separated from large HDP entities (like a macroglobulin), and HDP seems to be contaminated with some fraction(s) of HDL (Garbo, 1990; Kongshaug, 1993). Whilst, albumin and y-globulin are fairly well separated in gel elution chromatography (see Fig. 1 in Kongshaug, 1992) there is usually no such separation in ultracentrifugation (Chapman et al., 1981; Kongshaug et al., 1989; 1991). However, in a NaCl-KBr gradient a separation of the major classes of lipoproteins was achieved by the use of a swinging bucket rotor (SW 40) and, simultaneously, HDP entities were also partially separated into two or three groups (Kongshaug et al., 1990a,b). An aim of the present work is to identify the major HDP proteins that are responsible for the partial separation of HDP entities found by the use of the SW-40 swingout rotor. As well, this method is applied to the study of the binding of a tetrapyrrole, namely hematoporphyrin (HP), to human serum and human serum albumin (HSA). Separate centrifugation of HP-doped serum and HP-doped HSA should indicate whether in serum Hp is bound to HSA. An amphipatic dye, Hp is also one of the simplest tetrapyrroles and is a minor component of Photofrin II (which is widely used in clinical PCT). Loading of Hp to mouse LDL has been used for efficient tumor targeting of Hp to the MS-2 fibrosarcoma (Bare1 et af., 1986) and the binding of Hp to whole serum/plasma has been studied previously, both by gel elution chromatography (Jori et al., 1984; Jori, 1989) and by ultracentrifugation (Reyftman et al., 1984; Kessel, 1989; Kongshaug et al., 1989, 1990a,b; see Kongshaug, 1992). Gel elution (exclusion) chromatography has given a much lower percentage binding of Hp (and of Hp derivative, HpD) to lipoproteins in serum than has ultracentrifugation (see Kongshaug, 1992). Drug adsorption and the relatively poor separation noted of HDL from HDP in gel elution (cf. above) could be complicating factors (Kongshaug, 1992). Similarly, the use of a vertical tabletop rotor for the study of drug binding to serum/plasma led to substantial contami-

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nation of the HDL population by HDP entities (see Sykes et al., 1992). This high speed ultracentrifugation led to a much lower percentage binding to HDP than did lower speed ultracentrifugation (see Kongshaug, 1992). Although sera/plasma of different protein concentrations were employed in these studies, most of the differences found in the serum binding of Hp is probably due to methological problems (cf. Kongshaug, 1992, 1993). One methological question to be discussed herein is: does the type of rotor and/or salt gradient used affect the serum binding of Hp? To this end, the present results (serum; SW-40 swingout rotor; NaCl-KBr gradient) are compared with our previous results (plasma; 70.1 Ti fixed angle rotor; NaCl-CsCl gradient) (Kongshaug et al., 1989). The centrifugal fields of gravity (and hence the hydrostatic pressure) and spin times are substantially different for these rotors. This might affect lipoprotein structure to different extents. It has been suggested that the centrifugally generated hydrostatic pressure is the major factor behind lipoprotein instability during UC as any other cause(s) of such instability could not be identified (Kunitake and Kane, 1982). Another methological question to be discussed herein is a commonly overlooked challenge: protein-protein interactions and interaction between proteins and other substances may affect drug binding in serum so substantially that drug-protein association constants, determined for single proteins, may not apply to the drug-binding properties of same proteins in serum (cf. Kongshaug, 1992, 1993). Specifically, we discuss the present binding of Hp to serum proteins in light of published results for the binding of Hp to isolated HSA (Reddi et al., 1981; Smith and Neuschatz, 1983; Moan et al., 1985a,b; Rotenberg et al., 1987; see Table 1 in Kongshaug, 1992) and for isolated human LDL (Reyftman et al., 1984; Moan et al., 1985b). MATERIALS

AND METHODS

Chemicals

The materials were used as received. Hp (diHC1 salt) and human albumin (defatted; standard for electrophoresis) was obtained from Koch-Light Laboratories. Protosolv (detergent, containing Triton X-100 and several other substances) were from Porphyrin Products (a kind gift from Bruce Burnham). The dye stock

Hematoporphyrin

binding to human serum and albumin

313

Human blood was obtained from a healthy person (man, born 1942) after overnight fasting. The blood was clotted at room temperature (usually for 20-30 min) and serum was obtained by low speed centrifugation of the clotted blood. Apart from a few early samples, all our binding experiments performed to date have employed plasma/serum from this person. The present plasma/serum contained 38-40 mg/ml of albumin (normal range 35-50 mg/ml); 5.0-5.4 mmol/l of total cholesterol (normal range 48 mmol/l; includes both unesterified and esterified cholesterol), of which about 1.2 mmol/l was associated with HDL (normal range 0.7-l .9 mmol/l); and 0.860.88 mmol/l of triglycerids (normal range < 1.70 mmol/l) (Kongshaug, 1993). The use of plasma or serum from the same person guarantees an optimal reproducibility of the contents and composition of the blood proteins. Serum (0.2 ml) was incubated with or without dye for l-2 hr at 37°C before centrifugation. Most of the incubated serum samples were usually 100 or nearly 100% (> 97%) in serum.

1989) and may reduce possible effects on the lipoprotein composition during the centrifugation (cf. Kongshaug, 1993; Kongshaug et al., 1993). For these reasons we have also chosen to apply the sample at the top of the gradient instead of at d = 1.15 to 1.31 employed by the other groups referred to above. We usually apply a high salt density of 1.35 g/ml at the bottom of the centrifuge tube to obtain a good separation of HDP entities from the bottom wall. To our knowledge only Chung and colleagues (1986; see their Table 3) have previously employed such a high bottom density. Our gradients of type A contain the following volumes and densities: 3.3-3.5 ml of d = 1.006 g/ml; 3.5 ml of d = 1.063 g/ml; 4.5 ml of d = 1.21 g/ml; and 1.0 ml of d = 1.35 g/ml. In the much employed NaCl-CsCl type A gradient (Kongshaug et al., 1989, 1990a,b, 1991, 1993, 1995a,b; Kongshaug, 1992) the densities 2 1.063 are made up with CsCl (see Fig. 1 in Kongshaug et al., 1989) whilst in the present analogous gradient (NaCl-KBr type A gradient) these densities were made up with mixtures of NaCl and KBr according to Have1 et al. (1955). In contrast to the NaCl-CsCl A gradient, the NaCl-KBr gradient A does yield a partial separation of HDP entities (Kongshaug et al., work in progress). All the salt layers of our gradients are made up with 0.1 M Tris-HCl buffer, pH = 7.4 with 0.4 mg EDTA (potassium salt)/ml. Stock density solutions were carefully layered into the centrifuge tube by the use of a peristaltic pump, and the most dense solution was initially placed in the bottom of the centrifuge tube. The centrifuge tubes were of nontransparent allomer type.

Gradient

Centrifugation

Our salt gradients (Kongshaug et al., 1989, 1990a), as well as those used by others (Chapman et al., 1981; Kelley and Kruski, 1986), are somewhat modified versions of that used by Redgrave et al. (1975) the latter being a four step density gradient consisting of mixtures of NaCl and KBr. Like others (Redgrave et al., 1975; Foreman et al., 1977; Chapman et al., 1981; Nilsson et al., 1981) we always use 0.15 M NaCl in the top fraction of the gradient (d = 1.006 g/ml), but at higher densities we use in most of our gradients CsCl instead of NaCl/KBr or NaBr, since CsCl gives a lower molarity of salt at a given density. Such substitution may reduce the possibility for salting out of proteins and/or drugs (Kongshaug et al.,

The SW-40 rotor was employed at 30,000 to 32,000 rpm (geB< 130,OOOg) in Beckman centrifuges (L8-70M and/or L2-65B). The spin temperature was 15°C and the spin times were 6&68 hr. The rotor was allowed to decelerate/stop with the breaks off (usual practice by us and others). The present spin times seem to be sufficient for an acceptable separation of major proteins to be established. Thus, the 70.1 Ti rotor gave similar separation profiles of human plasma (in the NaCl-CsCl gradient type A) for spintimes 12-20 hr (gaff = 336,000 g), and for these conditions the product g, x spintime is less (by a factor of 1.2-l .9) than the corresponding product for the SW 40 rotor (for a 60 hr spin at 32,000 rpm; g,, = 130,000 g). By comparison,

solutions contained usually 0.625 mg dye/ml and were made up in aqueous alkaline sodium chloride. Human a -macroglobulin (minimum purity 90%) and human a,-antitrypsin (salt-free lyophilized powder) were Sigma products. Human gamma globulin (immunoglobulins) was from Kabi (165 mg/ml). Immunoglobulin M (isolated at our institute) was a kind gift from the immunological department. The salts (NaCl and KBr) were of the highest purity commercially available. Serum

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Chapman et al. (1981) using a NaCI-KBr gradient (roughly similar to the present gradient) in conjunction with the SW 41 Ti rotor (gcfl 197,300g) obtained a good separation of the lipoproteins with a spintime of 48 hr. The product g, x spintime in their experiment is only slightly higher (by a factor of about 1.1-1.3) than the values of this product in present conditions. Chapman er al. used a much larger volume of serum (3.5 ml serum in a gradient of 12.5 ml) than employed by us herein and previously (0.2-I .O ml serum/plasma in 12.5 ml). An equilibrium distribution of the proteins should be reached more rapidly in the latter conditions (owing to reduced viscosity and/or increased dilution of the proteins).

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escence from serum constituents was negligibly small as compared to the dye fluorescence. The contribution of serum components to the absorbance profile at 40.5 nm was accounted for (as was also done in our previous experiments). RESULTS

AND

DISCUSSION

The present curves (profiles) are measures of the distribution of proteins and dyes along the centrifuged gradient: the absorbance at 276 nm (proteins) or at 405 nm (HP), or fluorescence (Hp) is recorded for each of the volume fractions of the centrifuged volume. Each fraction is characterized by a fraction number (Figs I & 2). Increasing fraction numbers refer to increasing salt densities.

Analysis

The centrifuged gradient was analysed and fractionated from top downward by the use of a peristaltic pump and an LKB 2098 UVICORD III monitor. This allowed continuous reading of the absorbances at 276 nm (proteins) and 405 nm (dyes) (Kongshaug et al., 1989, 1990a). The fractions were collected and the dye fluorescence was measured in the various fractions (30 + 1 volume fractions of equal volumes). Inner filter effects were, in the fluorescence measurements, negligibly small, or made so by requisite dilution of the fractionated volumes (absorbance less than 0.1). In some experiments the fluorescence of the dyes were measured after addition of the liquid detergent protosolv (usually 10~1 protosolv per ml of undiluted or diluted fractions), or 1 N HCI, to the fractionated volumes. Typically 100-120 ~1 of each volume fraction was diluted with saline buffer or with 1 N HCI to 1.2 ml (0.1 - 1.2 ml). The saline-diluted fractions were either measured as such (1 N HCl) or 10 PI/ml of the detergent (protosolv) was added to each of the saline-diluted fractions before measurements (which began 2 hr or longer after the addition of protosolv). It was checked that the fluorescence was constant over a checked time-period of up to 26 hr. The HCl-diluted fractions were subject to heating at 90-95°C for some 30min. All fluorescence measurements were performed at (or very near to) the wavelengths of maximum excitation and maximum emission for the various conditions (A,, = 399 and E,,, = 421 in protosolv-containing solutions, and A,, = 403 nm and A,,,,= 595 nm in the HCl-containing fractions; whilst free Hp in saline buffer have A,, = 395 nm and &,, = 612 nm). The fluor-

A -

(Upper):

--

(Middle):

HSA+y-Globulin

--

(Lower):

ISM

-

Serum X5

: a-Antitrypsin - : a-Macroglobulin

-B -

(Upper):

HSA+y-Globulin : sum of (1) & (2)

-

(Lower): HSA (1) -- : y-Globulin

0

4

8

12

Fraction

(2)

16

20

24

28

number

Fig. 1. Absorbance at 276 nm vs fraction number in the centrifuged gradient (i.e. distribution of proteins along the gradient). A: Serum (0.2 ml; upper whole curve); HSA (42 mg/ml) plus I-globulin (13 mg/ml) (middle whole curve); immunoglobuhn M (0.94 mg/ml; lower whole curve); i-antitrypsin (2.6 mg/ml; pointed curve); and a-macroglobulin (0.24 mg/ml; stippled curve). B: HSA (42 mg/ml; curve I); 1-globulin (13 mg/ml; curve 2); the ordinates of curve 3 is the sum of the ordinates of curves 1 and 2. The incubated samples (0.2 ml) contained in most cases minor amounts of tetrapyrrohc dye (7-14 Gg/ml). The incubated serum samples were either 99% in serum (7pgdye/ml) or 100% (no dye). All samples contained proteins at their normal serum levels, the sole significant exception being amacroglobulin (whose concentration was 10 times less than its normal serum level). All concentrations given in this work refer to concentrations during the incubation before the centrifugation.

Hematoporphyrin

0

4

8

12

16

20

24

28

binding to human serum and albumin

2

Fraction number Fig. 2. A: 405 nm absorbance profiles of centrifuged free Hp (pointed curve) and of centrifuged Hp incubated with HSA (42 mg HSA/ml; stippled curve). After incubation protosolve (about 10 ,ul/ml) was added to the (undiluted) volume fractions and the fluorescence of Hp in these fractions was measured as described in the experimental section (whole curve; the measurements were performed after 2 days of storage at room temperature in the dark). The pointed curve (free Hp) has been normalized so as to have the same maximum ordinate as that of the whole curve. The absorbance and fluorescence curves for Hp plus HSA were normalized to the same peak value in the HSA-region of the gradient. B: dye profile (405 nm) of centrifuged Hp incubated with serum (whole curve) and HSA profile (276 nm) of HSA incubated with Hp (stippled line). These curves have been normalized to the same peak value in the HSA region. The intended dye concentration was (in these experiments) 7 pg Hp/ml in all experiments (both panels). Incidentally, the recovered amount of dye in the centrifuged gradient varied substantially in some of our experiments (from 612 fig HP/ml), partly because of inaccurate addition of small dye volumes (2.24~1 of the dye stock solution per 0.2 ml serum), and presumably one of the dye stock solutions was more concentrated than intended. The individual curves were therefore normalized to the same maximum ordinate value (in the HDL region) and thereafter the mean of the normalized curves were estimated (see shown error bars). In still another similar experiment (about 7 pg HP/ml), a similar distribution of serum-bound Hp was found (not shown).

Protein separation The protein distribution profile [Panel l(a)] is remarkable for its good resolution of the major lipoprotein classes and for the concomitant separation of HDP entities into two partially resolved peaks at high fns. VLDL, at the very top of the gradient (presumably fraction number region l-3), seems to be fairly well separated

315

from LDL (mainly found in fraction number region 3-8) and LDL from HDL. The minor hump in between the LDL and HDL distributions is probably lipoprotein (a) (Lp(a)) (to be checked in forthcoming experiments). There is some overlap between light HDL and Lp(a), and between LDL and Lp(a). Finally, HDL (fraction number region 8-20) seems to be satisfactorily separated from the major protein distribution (fraction number region 20-30) (in a previous report, the major HDL portion in the centrifuged NaCl-CsCl gradient type A was recentrifuged and found to be quite stable and apparently pure (it seemed to contain less than 5% of albumin, if any albumin at all; see Kongshaug et al., 1991). Overall, the present protein profile, Panel 1 (a), is fairly similar to the one previously found by us by the use of the present NaCl-KBr gradient A, employing a higher spin rate and lower temperature during the centrifugation (40,000 rpm; 7°C; 63 hr spin; see Panel 8 (c) in Kongshaug et al., 1990a). The previous profile was obtained in our initial experiments, and in this case blood from another blood donor was possibly used instead of the blood used thereafter by us for such studies. In the HDP region of the gradient the previous profile showed a middle peak flanked by two shoulders, in a roughly similar pattern as that presently found (a relatively low spin rate of 30,000-32,000 rpm was employed herein because of ageing of our rotor). These separation profiles offer a ready tool for obtaining a good overall picture of the pattern of drug distribution between the major groups of serum/plasma proteins, and represent the best such overall picture so far published in aqueous systems by single step separation procedures (ultracentrifugation, gel elution chromatography). Apparently, longer centrifugation (87 hr) resulted in a somewhat improved separation of albumin and y-globulin, but in this condition the latter protein was found largely together with the most heavy proteins (profile not shown). The lipoproteins and their densities could be affected by high salt concentrations under long-time centrifugations (see for example Kunitake and Kane, 1982; Kongshaug, 1993). However, only 5-10% of total apoA-I (the major apoprotein of HDL) was found outside the HDL region, i.e. in the very high density range 1.21-1.25 g/ml (Chapman et al., 1981). This could be due to some minor dissociation of apoA-I during the centrifugation or could mainly be components of native very high

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density lipoproteins (VHDL) (cf. Kongshaug, 1993). In contrast to us, Chapman er al. placed their sample initially at a high salt density which would favour lipoprotein instability. We have herein identified the major proteins behind the observed separation of HDP entities, namely albumin and y-globulin: a mixture of albumin and y-globulin (with concentrations similar to those in normal human serum) was centrifuged separately and the distribution profile was found to be similar to that of serum in the HDP region of the centrifuged gradient [Panel l(a)]. Moreover, separate centrifugations of HSA [curve 1 in Panel 1(b)] and y-globulin [curve 2 in Panel l(b)] showed that the peak at relatively low fraction number (23) was due mainly to albumin, whilst that at fraction number 27 was mainly due to y-globulin. In particular, the sum of the individual profiles of albumin and y-globulin [see upper whole curve in Panel l(b)] had a similar shape as that of centrifuged serum and of the centrifuged mixture of albumin and y-globulin [see Panel l(a)]. The present serum contained about 40 mg HSA/ml (as did the present HSA-containing samples), whilst the serum concentration of y-globulin has not yet been determined. Presumably, the present serum contained about 13 mg/ml (normal serum concentration of y-globulin) as did our y-globulincontaining solutions. Even so, the absorbance of the mixture of albumin and globulin was substantially lower than that of the HDP entities in serum [see Panel l(a)]. This is so because the HDP proteins include a number of other proteins that are separated together with HSA and y-globulin and, additionally, the optical extinction coefficients (at 276 nm) of purified HSA and purified y-globulin could be lower than those of HSA and y-globulin when present in serum (because of their interactions with serum components; HSA, for example, may interact with fatty acids, amino acids and bilirubin, and these and other interactions may affect the optical properties of HSA). Admittedly, there is a substantial overlap of the albumin and y-globulin profiles, particularly around fraction number 26 [cf. panel (b)]. Even so, the present ultracentrifugation can easily be applied to decide whether a given dye may bind to albumin or to y-globulin. A complicating factor is that other proteins may distribute themselves in similar manners as albumin or y-globulin. In the present work, cl,-antitrypsin (at its normal blood concentration; no serum

Moan

present) was distributed slightly to the left (peak at fraction number 22) of albumin, whilst the separately obtained profiles of immunoglobulin M (IgM) and CY -macroglobulin showed increasing concentrations towards the very bottom of the tube [Panel l(a)], whereto also fibronectin migrates (not shown). The shown profiles refer to the normal blood concentrations of these proteins, except for a-macroglobulin whose concentration was (in this experiment) only 10% of its normal blood concentration. There are four keys to the understanding of the present partial separation of high density proteins (HDP). Firstly, the type of gradient used: no obvious separation occurs in the NaCl-CsCl gradient type A under otherwise similar conditions (Kongshaug, work in progress). So high salt concentrations may affect the hydrated densities of one or more HDP entities in manners that depend on the type of salt. Secondly, optimal separation may occur when the sample is initially placed in the top layer of the gradient, whilst the separation in the HDP region is less satisfactory when the sample is placed at high salt density (Kongshaug et al., 1990a). The third key is the relatively low amount of serum applied, viz. only about 0.2 ml in a total volume of about 12.5 ml, implying considerable dilution of the serum/plasma during the centrifugation. The significance of the degree of serum/plasma dilution (prior to the centrifugation) upon the separation after centrifugation will be illuminated in a separate contribution (Kongshaug, 1992, 1993; Kongshaug, work in progress). Finally, the use of the swingout bucket is a prerequisite to optimal separation in the HDP region of the gradient (Kongshaug, work in progress). This is consistent with the experience that the use of high speed vertical or nearly vertical rotors (fixed angle rotors) tends to give a somewhat less satisfactory separation of serum/plasma proteins than does the application of swingout rotors (cf. Chung et al., 1986). Thus, in the former rotors the gradient is switched during and after the centrifugation: whilst before (and after) the centrifugation the density varies along the centrifugation tube, the variation in density is perpendicular to the length-direction of the tube during the centrifugation in a vertical rotor (see Chung et al., 1986). The switching-back of the gradient during the deceleration of the spinning rotor probably reduces somewhat the quality of the separation.

Hematoporphyrin binding to human serumand albumin

311

increased at increasing salt concentrations. Centrifugation of free Hp distributed this dye However, in serum there is no evidence that the mainly in the upper part of the gradient: the binding of Hp to HDP entities depends substantially on the salt concentration (see below). The 405 nm absorbance profile was fairly symmetric around the position that the dye had before the simplest explanation of the profile of free Hp centrifugation [pointed curve in Panel 2(a)]. (HSA present) is that the sample was dragged along with albumin during the early stage of Thus, this dye moved during the centrifugation centrifugation, i.e. while the sample remained according to ordinary diffusion (determined by relatively undiluted and hence had a relatively the dye concentration gradient). This also holds large viscosity. The free/unbound Hp moved for Sn-etiopurpurin (Kongshaug et al., 1993). thereafter (during most of the centrifugation) by As the migration of these dyes was not affected ordinary diffusion (as did free Hp in the absence significantly by the field of gravity during the of HSA). Additionally, there was probably a centrifugation, these dyes did not tend to form gradual and increasing dissociation of HSAhigh density dye aggregates (dense aggregates) bound Hp during the centrifugation, giving rise in saline buffer. This contrasts with the beto a tail of Hp behind the HSA-bound Hp (such haviour of the more hydrophobic etiopurpurin a tail would grow wider with increasing fraction (Kongshaug et al., 1995a,b). number). The observed profile of free Hp is thus considered to be the composite result of these Binding of Hp to human serum albumin two processes. Centrifugation of HSA doped with Hp led to The dye fluorescence profile of undiluted fraca bimodal 405 nm absorbance profile of Hp tions containing, however, the detergent protoalong the gradient: the peak at high fraction solv [whole curve in Panel 2 (a); I,, = 401 nm; numbers (2&30) represents Hp bound to HSA, A,,, = 621 nm; cf. experimental] was similar in whilst the peak at low fraction numbers (l-20) form to (i) the corresponding profile of highly is free HP [stippled curve in Panel 2(a)]. Some diluted fractions (not shown; dilution reduced of the HSA-bound Hp probably dissociated the concentration of salt and also reduced the during the downward migration of HSA (see variation in such concentration in the gradient), below). and (ii) the fluorescence profile for the highly To separate unbound Hp from HSA-bound diluted HCl-containing fractions (not shown). Hp, it is decisive to position the sample in the This latter profile was measured after heating at top salt layer of the centrifuge tube prior to 9&95”C for 1 hr; (&,, = 403 nm; 1, = 596 nm). ultracentrifugation. Then, during centrifuHeating in HCl destroys the proteins and thus gation, free Hp remains largely in the upper part fully extracts Hp, whilst the high and uniform of the centrifuge tube (since the field of gravity concentration of HCl (1 N) in highly diluted does not influence its motion), whilst HSAfractions (cf. experimental section) yields a relabound Hp migrates toward the lower end of the tively constant ionic mileu along the gradient tube. To our knowledge these are pioneering (which guarantees a constant Hp fluorescence experiments. quantum yield). Moreover, such heating would The dye peak at low fraction numbers (HSA hydrolyze esterified dye impurities, including present) was shifted towards higher fraction oligomers linked by ester bridges, if such were numbers relative to that of free Hp as observed present. Thus, the similarity of the three fluorin the absence of HSA [cf. 405 nm profiles in escence profiles indicates that they are all Panel 2(a)]. It might be thought that the peak in good measures of the distribution of total the free Hp distribution (HSA present) arose in (monomeric plus aggregated molecules) of Hp the following manner: as more and more Hp throughout the gradient. was dissociated from HSA during the downThe absorbance pattern of HSA-bound Hp ward migration of the HSA-Hp complex, the (undiluted fractions; no protosolv) was similar rate of dissociation decreased, possibly as a to the fluorescence pattern of monomeric or result of the decreasing concentration of HSA- monomerized free Hp in the HSA region [cf. see bound Hp (cf. law of mass action). However, peaks at high fraction numbers in Panel 2(a)]. there is an increasing dilution of HSA-bound This indicates that Hp was bound to HSA Hp during its downward migration and this as mainly monomeric Hp in the undiluted gradiwould favour dissociation of Hp. Alternatively, ent. The ratio of dye fluorescence to dye abthe strength of the binding of Hp to HSA sorbance was larger in the LDL and HDL Free Hp (no protein present)

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Kongshaug

regions than in the HDP region [Fig. 2 (a)]. This probably means that free Hp was somewhat aggregated in the undiluted gradient. Thus, aggregated Hp has a lower extinction coefficient at 405 nm than has monomeric Hp (cf. Beltramini et al., 1987) and aggregated Hp either does not fluoresce or it fluoresces much less than monomeric Hp. The area of the fluorescence peak around fraction number 13 (fraction number region I-20, corresponding to unbound Hp) divided by the area under the whole fluorescence curve (sum of unbound and HSA-bound Hp) is equal to 0.75. So 75% of Hp was in the free form after centrifugation of HP-doped HSA. This percentage was substantially larger than the corresponding percentage (64%) for the 405 nm absorbance profile (cf. Panel 2(a), the difference between these percentages being due to partial aggregation of free Hp (cf. above). Several groups have studied the binding of Hp to HSA by fluorometric methods (no ultracentrifugation): it has been found that HSA has a high affinity binding-site for Hp (one site per HSA molecule) with association constant of the order of 106/M (see Table 1 in Kongshaug, 1992). One would expect therefore that during incubation (600 PM HSA) there would be some 600 HSA-Hp complexes per free Hp. There occurs a substantial dilution of HSA during the centrifugation (on the average, by about a factor of ten; see, in Panel 1 (b), the profile of HSA in the centrifuged gradient). Even so, the population of free Hp observed after centrifugation in the HSA-containing sample is far larger than would be expected on the assumptions that (i) the HSA-Hp complexes moved together with the bulk of uncomplexed HSA, and (ii) the association constant for the Hp binding to HSA is of the order of 106/M. Thus, the present HSA (electrophoretically pure) had a relatively low affinity for Hp in the gradient. Assuming that the ratio of unbound Hp to that of HSA-bound Hp was the same after centrifugation as before centrifugation, it follows that the association constant for the binding of Hp to HSA (Hp + HSA P HSA-Hp) would be 1 x 103/M (for one binding site per HSA molecule). This estimate is of correct order of magnitude, yet is a rough one because, as noted above, some Hp was dissociated along the gradient during the centrifugation. Hardly more than one third of the free Hp was possibly formed by such dissociation [see the form of stippled curve in panel 2(a) and imagine that the free Hp is super-

and Johan

Moan

imposed upon a tail of dissociated Hp. a tail that grows wider with increasing fraction number]. If so, the present estimate is correct within a factor of two. Apparently, previous studies of the binding of Hp with HSA employed more impure HSA batches. Binding of drugs to HSA may depend to some extent on the conditions of isolation and/or storage of HSA, as such conditions may affect the purity and/or binding properties of HSA (Kongshaug, 1992). For example, commercial Cohn fraction V albumin contains impurities with absorbance around 400 nm (these “coloured” impurities absorb 1.5-3 times more light at 400 nm than does chromatographically purified (electrophorectitally pure) albumin) (see Fig. 6 and Table 1 in Bergliif et al., 1983). However, the binding of deuteroporphyrin to HSA was the same for HSA purified by affinity chromatography, for Cohn fractionated HSA (defatted HSA and fatty-acid-containing HSA), and for pure monomer HSA (Moehring ef al., 1983; Rotenberg et al., 1987). The association constants for the binding of Hp to HSA were roughly similar for Cohn fraction V HSA (more or less fatty-acid carrying batch from Sigma) (Reddi et al., 1981) for Cohn fraction V defatted HSA (Sigma) (Rotenberg et al., 1987) and for freeze-dried HSA (Kabi AB product) (Smith and Neuschatz, 1983). Binding qf’ Hp to serum

The 405 nm absorbance profile of serumbound Hp is shown in Panel 2(b). Also shown in this panel is the 276 nm profile of HSA (obtained separately). This HSA profile was normalized to the peak of the Hp pattern in the HDP region. At high fraction numbers these patterns coincide [see Panel 2(b)]. Such a similarity in the dye and HSA profiles suggests that the binding of Hp to the HDP entities in serum was binding mainly or solely to HSA. There seems thus to be little or no binding of Hp to y-globulin. The percentage binding of Hp among LDL, HDL and HDP has been obtained from the areas under the LDL, HDL and HDP regions, respectively, both absorption profiles [Panel 2(b)] and fluorescence profiles (not shown). Great care has been taken to ascertain that there is no effect of the gradient salt upon our measurements (see below). The results are summarized in Table 1 which also includes previous results obtained by the fixed angle 70.1 Ti rotor and the NaCl-CsCl A gradient.

Hematoporphyrin

379

binding to human serum and albumin

Table 1. Distribution

of Hp among serum proteins Percentage distribution”

Method SW 40/Absorp 614pug HP/ml SW 4O/Fluor(protos) 7 pegQ-W SW 40/Fluor(HCl) 7 wz HP/ml SW 40/Absorp 70 p g HP/ml SW 40/Fluorb 70 pg HP/ml 70.1 Ti/Absorp’ 14-70 pg HP/ml

LDL

HDL

HDL

(n)

12.4 + I.5

53.5 f. 0.6

34.0 & 1.9

(3)

12.4 + 1.9

59.6 & 1.4

21.9 + 1.7

(5)

11.3 + 1.0

61.2 k 2.6

27.5 + 3.1

c-4

7.7 f 0.1

58.8 + 2.8

34.2 + 2.8

(2)

8.3 + 0.7

63.5 + 3.5

28.2 + 4.2

(2)

lo+ I

55 + 4

34+ 3

(3)

“The percentage distribution is obtained from the areas under various protein regions. These regions are (SW 40/NaCl-KBr gradient) fraction numbers l-8 (LDL), 8-20 (HDL) and 20-30 (HDP); the corresponding regions for the 70.1 Ti rotor (NaCl-CsCi) being I-10, l&22 and 22-30, respectively (cf. Kongshaug et al., 1989). The errors are + one standard deviation (n > 3) and + one deviation from the mean (n = 2). Absorp refers to 405 nm absorption profile, Fluor(protos) and Fluor(HC1) to samples containing protosolv (10 nl/ml) or 1 N HCl (see experimental). bMean of two exueriments measuring Fluor(protos) and Fluor(HCl), respectively. cResults from Kongshaug et al., 1989.

At low dye concentration (6-12 pg Hp per ml concentration was reduced from 70 to 7 pg Hp of serum; mean concentration about 7 pg per ml of serum. Thus the percentage binding to HP/ml) Hp was considerably bound to LDL LDL was, seemingly, as much as 1.6 times (12.4 + 1.5% of the amount of Hp in the gradihigher at the low than at the high dye concenent), but mainly to HDL (53.5 + 0.6%) and tration. This was confirmed by fluorescence substantially to HDP (34.0 + 1.9O/,) (cf. Panel measurements: (8.3 + 0.7) % binding of Hp to 2(b) and Table 1; this panel refers to two out of LDL, (63.5 k3.5) % to HDL and (28.2k4.2) three experiments; small deviations in the forms % to HDP (mean of two experiments, referring of the profiles may yield relatively large errors to the presence of protosolv and HCl, respectin some ordinate values, yet the areas under the ively). These percentages are closely similar to individual profiles may be similar). The minor those obtained by us previously, by the use of binding to VLDL has been included in the plasma samples in conjunction with another percentage for the LDL binding. Thus, the area rotor (fixed angle 70.1 Ti) and the NaCl-CsCl under the serum-bound profile in the fraction gradient A: 10 f 1% (LDL), 55 + 4% (HDL) number region l-3 was only 1% of the total and 34 + 3% (HDP) (see Table 1 in Kongshaug area and only 7% of the area in the fraction et al., 1989; present Table 1). The latter results number region l-8 [cf. Panel 2(b)]. Apparently, were average results found for Hp concenmaximum 1% of total binding and 7% of the trations in the region 14-70 pg Hp per ml of binding in the fraction number region l-8 was plasma. due therefore to VLDL-binding of Hp. MorePlasma and serum behaved, then, in similar over, the low binding of Hp to Lp(a) [cf. Panel manners with respect to Hp binding, and neither 2(b)] is included in the percentage for HDL the type of salt gradient nor the type of rotors binding. employed seem to affect significantly the bindAt 70 pg Hp per ml of serum, the present ing pattern of Hp among the blood proteins. percentage binding was, according to dye ab- Salts commonly used in density gradients (KBr, sorbance profiles (mean of two independent NaBr) may affect to some extent certain blood experiments; profiles not shown): 7.7 + 0.1 proteins and/or their binding of some types of (LDL), 58.8 f 2.8 (HDL) and 34.2 rf: 2.8 drugs, such as tetraphenylporphines and per(HDP). Apparently, the percentage binding to haps other substances as well (Kongshaug, LDL increased by 4%, whilst the percentage 1992, 1993). Such effects include minor precipibinding to HDL decreased by 4%, as the dye ,tation of proteins or protein components in

380

Magne Kongshaug and Johan Moan

NaBr gradients (see p. 1352, Groot et al., 1982) and/or of protein-drug complexes in NaBr or NaCl/KBr gradients (Kongshaug, 1992, 1993). These effects are more pronounced when plasma, rather than serum, is used. They seem to be related therefore to the blood coagulation system (Kongshaug, 1992, 1993). Actually, such effects seem to be small or absent in serum (Kongshaug, 1992, 1993). In particular, serum should be used in conjunction with salts such as NaBr and KBr (and tetraphenylporphine drugs). This is why the present study involves serum (rather than plasma). With respect to possible systematic errors, how accurate are the just given present values for the percentage binding of Hp to serum proteins, as obtained by absorbance measurements? The estimated percentages rest on the assumption that the extinction coefficient for Hp is constant throughout the gradient. The adequacy, or otherwise, of the present results was further investigated by fluorescence measurements of diluted fractions containing either the detergent protosolv or fairly concentrated HCl (see Analysis). In five separate centrifugations, fluorescence measurements of saline-diluted volume fractions containing protosolv (cf. experimental) gave the following results for the average percentage binding (Table 1): (12.4 + 1.9) % of Hp was bound to LDL, (59.6 & 1.4) % to HDL and (27.9 rfI 1.7) % to HDP (concentrations ranged from about 3.5-12 pg HP/ml, the mean concentration being near to 7 p g/ml). The results found after heating in HCl were closely similar (mean of two independent centrifugations): 11.3 + 1.O% (LDL), 61.2+2.6% (HDL) and 27.5 + 3.7% (HDP). As explained in the previous section (bottom p. 13), there is no effect of the gradient salt upon the measured fluorescence in these conditions. Even so, these results are similar to those determined by absorbance measurements on undiluted fractions (cf. above). In more detail, however, the fluorescence measurements gave a slightly higher binding to HDL and a correspondingly lower binding to HDP than did the absorbance measurements. This minor difference suggests that Hp had somewhat different extinction coefficients at 405 nm, in manners that were dependent upon the type of protein. Specifically, assuming that the extinction coefficients for Hp binding to HDL and HDP were about a factor of 0.85 lower and a factor of 1.15 higher, respectively, than that for Hp binding to LDL, then the absorbance and fluorescence

measurements would agree. Moreover, the similarity between the dye absorption and fluorescence profiles suggest that the dye was bound mainly as monomeric Hp; the minor variation in the extinction coefficient could (but need not) imply some minor aggregation of Hp in the HDP region. A possible dissociation of some Hp from HSA during the centrifugation (serum present) could imply that the found “binding” of Hp to LDL and HDL is somewhat larger than the actual binding in uncentrifuged serum. Conversely, if the sample had been positioned prior to the centrifugation at a high fraction number near to the bottom of the centrifuge tube (in the HDP region), then after centrifugation the percentage of Hp apparently bound to HDP might be somewhat larger than the corresponding percentage found for an initial sample position at low salt density (since the HSA-bound Hp would remain near to HSA, and since some Hp may dissociate from the lipoproteins in the HDP region during their upward migration and would remain where formed (apart from diffusion). In fact, placing their sample initially at a high salt strength (1.21 g/ml) in a NaCl-KBr gradient in a swinging bucket rotor, Reyftman et al. (1984) obtained (by fluorescence measurements) a larger percentage binding to HDP (51-55%) than the corresponding present value (28%). However, this difference need not be due entirely to dissociation of HSA-bound Hp during the centrifugation. Alternatively, the serum used by Reyftman et al. (1984) possibly contained more HDP than did the present serum. Actually, during migration of HSAbound Hp (during the spin) there was, in plasma-containing samples, little or no dissociation of Hp: when plasma incubated with Hp was placed initially at a high salt density and at a low salt density, respectively, the percentage binding of Hp to HDP was only marginally larger (by a factor of 1.1) in the former than in the latter position (see Table 3 and footnote a 1992). Referring to the in Kongshaug, NaCl-CsCl gradient A and the 70.1 Ti rotor, this latter result also suggests that the exposing of the samples initially to widely different salt concentrations resulted in only minor (although perhaps significant) effects on the distribution patterns of Hp among the plasma proteins. Similarly, we have found that exposing uncentrifuged samples (Hp plus plasma) containing increasing concentrations of salt (CsCl) does not affect significantly the dye absorbance or fluor-

Hematoporphyrin

escence (Kongshaug, 1992; Kongshaug et al., 1993). The relatively high binding of Hp to LDL at low dye concentration can be understood if LDLs contain high affinity binding sites for Hp with a relatively low capacity for Hp binding (high-affinity-low-capacity binding sites; see further below). A concentration of 7 pg Hp per ml serum corresponds to about 10 PM. Since about 10% of Hp was bound to LDL, this means that at this concentration about one Hp molecule is bound per LDL particle [since in normal human serum the (normal) concentration of LDL is about 1 PM; see, e.g. Kongshaug et al., 19931. As in normal human serum the concentration of HDL is about 13 PM, only each third HDL particle contained, on the average, one Hp molecule at 7 pg HP/ml, and similarly only at most one of 200 HSA molecules contained one Hp molecule. Thus, at low Hp concentration (7 pg/ml) we are almost certainly dealing essentially with the binding of monomeric Hp to LDL, HDL and HSA in human serum/plasma. The binding of one Hp molecule per LDL might decrease the affinity for one or more other Hp molecules. Obviously, since Hp was bound extensively to HDL even at low dye concentration, there also exists one or more binding sites per HDL particle. However, the high affinity sites on LDL may have 2-3 times as large an association constant for Hp than has the binding sites on HDL for Hp. To see this, let [Hp-HDL] denote the concentration of HDL-bound Hp in the incubation volume, the corresponding symbol for LDL binding being [Hp-LDL]. Similarly, [LDL] and [HDL] are concentrations of LDL and HDL, respectively, whilst KLDL and KHDL are association constants for the binding of Hp to LDL and HDL, respectively, and let nLDL and nHnL signify the average number of binding sites per LDL and per HDL particle, respectively. Application of the law of mass action to the binding of Hp with LDL (Hp + LDLF? Hp-LDL) and to HDL (Hp+HDLs Hp-HDL), and elimination of the common parameter (the concentration of free Hp) from the two equilibrium expressions, yields: nLDL

x

KLDL

=

nHDL

x KHDL

[HDL] [Hp-LDL] x [LDLI ’ [Hp-HDL]

381

binding to human Serum and albumin

[‘I Thus, putting the HDL to LDL concentration ratio equal to 13 (see above) and the

LDL-bound Hp to the HDL-bound Hp ratio equal to the present experimental value of 0.20 (see values for percentage binding given above), then: nLDL

x KLDL

=

2.6

x

nHDL

x

&DL

The effective association constant (n x K) was thus 2.6 times larger for LDL than for HDL in our conditions at low Hp concentrations. By comparison, it may be similarly shown that at 70 pg HP/ml the effective association constant for LDL binding was 1.7 times larger than that for HDL binding. Since at 7 ,ug HP/ml there was only one Hp molecule per LDL particle, on the average (cf. above), there were about 7 Hp molecules bound per LDL at the high dye concentration (70 pg HP/ml). The seven binding sites on each LDL that were occupied by Hp (in serum) at 70 pg HP/ml had, then, seemingly a lower average association constant than had the single Hp molecule per LDL at 7 ,ug HP/ml. The number of binding sites (n) and the association constant (K) should be considered as average quantities for each protein and for the specified (range of dye) concentration(s) investigated. Apparently, the binding sites on LDL were saturated at the high dye concentration (seven sites occupied by Hp). Thus, fluorometry in conjunction with dialysis and Scatchard plotting, gave an average number of 7 binding sites for Hp per human LDL (isolated) and an average association constant of 0.9 x 106/M (Reyftman et al., 1984). Spectroscopic evidence suggests that Hp binds to apoB (possibly to tryptophyl residues, each LDL containing about 13 such residues) (Moan et al., 1985b), but all such residues may not be available to Hp. The availability of binding sites could depend on the presence of other proteins or substances (cf. below). Still other types of binding sites could exist on LDL and HDL (possibly in a mileu-dependent manner). The binding sites would be increasingly occupied at increasing Hp to serum concentration ratio. At high concentration Hp could be partitioned into certain hydrophobic/lipid regions of LDL (cf. Beltramini et al., 1987). Plasma proteins could interact in manners that affect binding sites (for some drugs) and, in particular, HDP deficient plasma/serum may show a different distribution of a given dye among the lipoproteins than does whole serum/ plasma (for a previously discussed possible example involving benzoporphyrin derivative, see Kongshaug, 1992). Moreover, the solubility

382

Magne

Kongshaug

(due to protein binding) of the carcinogen benzo (a)pyrene-7,8-dihydrol in plasma was twice as high as the sum of the solubilities of this drug in separately incubated lipoprotein- and nonlipoprotein fractions (Shu and Nichols, 1981). Additionally, let us assume that the association constants for HP-binding to isolated (Cohn V fractionated) HSA and to isolated human LDL apply to the situation in human serum. Referring, then, to the absence of other proteins, these association constants have magnitudes of KHsA= (l-4) x 106/M (see Table 1 in Kongshaug, 1992) and KLoL = 0.9 x 106/M (see above). Similarly, the number of binding sites(regions) per protein molecule are nLDL = 7 and nHSA= 1, whilst normal concentrations in human serum are 1 p M (LDL) and 600 PM (HSA). These values may be used to estimate the ratio of HSA-bound to LDL-bound Hp from the following expression (cf. equation

PI): [HP [HP -

HSAI=-x----xtlHSA&A LW VLDL KLDL

[HSAI P-W [21

This ratio is equal to 95 (KHSA= 1 x 106/M) or 380 (KHSA= 4 x 106/M). By comparison, the present experimental value is 4. Using for serum K LnL = 0.9 x 106/M and nLbr = 7, and using the experimental value of 4 for the HSA-bound to LDL-bound Hp concentration ratio, equation [2] yields KHsA= 4 x 104/M (for nnsA = 1). This value is about two orders of magnitude lower than that found experimentally for isolated Cohn fraction V HSA and 40 times larger than that found herein for electrophoretically pure HSA. Thus, if HSA in human serum would bind Hp substantially stronger than the present defatted HSA (but substantially less strongly than Cohn-fraction V HSA), then there need be no discrepancy between expected and observed binding to LDL in human serum. Alternatively, it could be assumed that KHsA= 106/M in serum. If so, according to equation [2] the product nLDL x KLDL would be as large as 1.5 x lO*M in order to satisfy the present experiments. The causes behind the difference between the estimated and experimental values for the LDL-Hp to HSA-Hp ratio in serum are presently not understood. We have assumed above that the available parameters for Hp binding to isolated LDL and HSA were.appropriately determined. In particular, we have

and Johan

Moan

assumed that impurities in the Hp products (which contain 70-90% Hp,) did not seriously affect the published equilibrium constants for the binding of Hp with LDL and HSA. If so, it then seems that interactions between plasma proteins affect their binding with Hp (such interactions are nonexistent in pure isolated proteins). In particular, HSA in serum might be associated with components that separates with HSA during Cohn fractionation (cf. previous section). Cohn fractionated albumin is electrophoretically quite impure (cf. Fig. 3 in Eriksson et al., 1983) and especially contains both c1-and /3-globulins (see Fig. 16, p. 15, in Curling, 1983). Containing a variety of different proteins, these globulins migrate, in electrophoresis, to the same positions as do HDL (a-globulins) and LDL (p-globulins) (cf p. 18 in Putman, 1975). Hetero complexes between albumin and lipoproteins might thus form in serum, each such complex containing one or more albumins and, if so, such complexes may exhibit high affinities for drugs such as Hp and HpD. It is even conceivable that certain LDL-Hp complexes and/or HDL-Hp complexes could be bridged to HDP entities via the dye (Kongshaug, 1993). If so, such complexes might dissociate to different degrees during gel elution chromatography and ultracentrifugation and such possible phenomena might contribute to the herein noted different serum binding of Hp found by gel elution, low speed ultracentrifugation and high speed ultracentrifugation. Possibly, like Shu and Nichols (1981) we have been glimpsing above into a new field of molecular biology: interactions/associations between different proteins (which might occur in vitro and in viva) and the effect of such interactions on drug binding; the use of separation methods may significantly interfere with the phenomena we are trying to study. The present achievement of a single-spin separation of the major lipoprotein groups and of HDP groups, as well as the other aspects of the present approach discussed above, sets a useful and broad stage for further developments (cf. Kongshaug, 1993). Several other photosensitizers (Photofrin II, chlorin es, tetraphenylporphine tetrasulfonate and protoporphyrin) have already been studied by the present experimental approach (Kongshaug and Moan, work in progress). Acknowledgement-We drawing of the figures.

are indebted

to Aasa

Dalen

for her

Hematoporphyrin

binding to human serum and albumin

Beltramini M., Firey P. A., Ricchelli R., Rodgers M. A. J. and Jori G. (1987) Steady-state and time-resolved spectroscopic studies on the hematoporphyrin-lipoprotein complex. Bichemistry 26, 68526858. Bare1 A., Jori G., Perin A., Pagnan A. and Biffanti S. (1986) Role of high- low- and very low-density lipoproteins in the transport and tumor-delivery of hematoporphyrin in vivo. Cancer Lett. 32, 145-150. Berglof J. H., Eriksson S., Suomela H. and Curling J. M. (1983). In Separation of plasma proteins (Edited by Curling J. M.) pp. 51-58. Pharmacia Fine Chemicals AB, Uppsala, Sweden. Chapman M. J., Goldstein S., Lagrange D. and Laplaud M. (1981) A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classesfrom human serum. J. Lipid Res. 22, 339-358. Chung B. H., Segrest J. P., Ray M. J., Brunzell J. D., Hokanson J. E., Krauss R. M., Beaudrie K. and Coni J. T. (1986) Single vertical spin density gradient ultracentrifugation. In Methods in Enzymology (Edited by Segrest J. P. and Albers J. J.), Vol. 128, pp. 181-209. Academic Press Inc., New York. Curling J. M. (1983) Current practice and future possibilities in plasma protein fractionation. In Separation of plasma proteins (Edited by Curling J. M.) pp. 5-33. Pharmacia Fine Chemicals AB, Uppsala, Sweden. Dougherty T. J and Marcus S. L. (1992) Feature Article. Therapy.

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Biology and Clinical Use (Edited by Bock G. and Harnett S.) pp. 90. Ciba Foundation Symposium 146. Wiley, Chichester. Kessel D. and Woodburn K. (1993) Biodistribution of photosensitizing agents. Int. J. Biochem. 25, 1377-1383. Kessel D., Dougherty T. J. and Chang C. K. (1991) Photosensitization by synthetic diporphyrins and dichlorins in vivo and in vitro. Photochem. Photobiol. 53, 475479. Kongshaug M. (1992) Distribution of tetrapyrrole photosensitizers among human plasma proteins. Int. J. Biothem. 24, 1239-1265. Kongshaug M. (1993) Binding of tetrapyrrole photosensitizers to human plasma proteins, studied by ultracentrifugation. Thesis. Oslo University. Kongshaug M., Moan J. and Brown. (1989) The distribution of porphyrins with differnt tumor localising ability among human plasma proteins. Er. J. Cancer 59, 184-188. Kongshaug M., Moan J., Rimington C. and Evensen J. (1990a) Binding of PDT photosensitizers to human plasma studied by ultracentrifugation. In Photodynamic Therapy of Neoplastic Disease 5, (Edited by Kessel D.) Vol. II, pp. 4362. CRC Press, New York. Kongshaug M., Rimington C., Evensen J. F., Peng. Q. and Moan J. (1990b) Hematoporphyrin diethers-V. Plasma protein binding and photosensitizing efficiency. Int. J. Biochem. 22, 1127-1131. Kongshaug M., Cheng L.-S., Moan J. and Rimington C. (1991) Interaction of cremophor EL with human plasma. Chemistry,

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Kongshaug M., Moan J., Cheng L.-S., Garbo G., Kolboe S., Morgan A. R. and Rimington Claude (1993) Binding of drugs to human plasma proteins, exemplified by Sn(IV)-etiopurpurin dichloride delivered in cremophor and DMSO. Int. J. Biochem. 25, 739-760. Kongshaug M., Cheng L.-S., Moan J. and Morgan A. R. (1995a) Binding of etiopurpurin and tin-coordinated etiopurpurin to human plasma proteins. Delivery in cremophore EL and dimethyl sulfoxide (paper II). Int. J. Biochem. Cell Biol. 27, 71-76. Kongshaug M., Moan J., Cheng L.-S. and Morgan A. R. (1995b) Binding to etiopurpurin to human plasma proteins. Delivery in cremophore EL and dimethyl sulfoxide (paper III). Int. J. Biochem. Cell Biol. In press. Kunitake S. T. and Kane J. P. (1982) Factors affecting the integrity of high density lipoproteins in the ultracentrifuge. J. Lipid Res. 23, 936940. Mazitre J. C., Morliere P. and Santus R. (1991) The role of the low density lipoprotein receptor pathway in the delivery of lipophilic photosensitizers in the photodynamic therapy of tumors. J. Photochem. Photobiol. B: Biol. 8, 351-360. Moan J. and Berg K. (1992) Photochemotherapy of cancer: experimental research. Photochem. Photobiol. 55, 931-948. Moan J., Rimington C. and Western A. (1985a) The binding of dihematoporphyrin ether (photofrin II) to human serum albumin. Clinica Chimica Acta 145, 227-236. Moan J., Rimington C., Evensen J. F. and Western A. (1985b) Binding of porphyrin to serum proteins. In Methods in Porphyrin Photosensitization (Edited by Kessel D.), pp. 193-205. Plenum Press, New York and London. Moehring G. A., Chu A. H., Kurlansik L. and William T. J. (1983) Heterogeneity of albumin as detected by its

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