The effect of different surface chemical groups on drug binding to liposomes

Chemistry and Physics of Lipid.s, 34 (1983) 81-92 Elsevier Scientific Publishers Ireland Ltd.

THE EFFECT OF DIFFERENT SURFACE CHEMICAL DRUG BINDING TO L I P O S O M E S

81

G R O U P S ON

PETER SCHLIEPERa and RUDOLF STEINERb

aInstitut [iir Pharmakologie and blnstitut fiir Klinische Physiologie, Universitiit Diisseldorf, Moorenstrasse 5, D-4000 Diisseldorf (Federal Republic of Germany) Received March 5th, 1983

accepted May 20th, 1983

The binding of four secondary and tertiary amine drugs with local anesthetic activity (propranolol, tetracaine, lidocaine, procaine) to liposomes containing charged surface groups of different chemical composition has been investigated. Binding is determined by measurement of partition coefficients and of drug induced zeta potential changes of the liposomes. For propranolol 30% of the total amount of drug dissolved in phosphatidylcholine is located as protonated form in the liposome surface. Fifty percent of tetracaine and 13% of procaine contribute to the surface charges. Negative surface charges (phosphatidylserine) facilitate drug binding and drug protonization in the liposome surface. Positive surface charges (hexadecyltrimethylammonium) prevent the protonization of the drugs. Different chemical groups of single negatively charged phospholipids or of electrostatically neutral lipids have no significant effect on drug binding which proves that binding is not influenced by steric and bulky head group configurations. The drugs interact hydrophobically with the lipid phase in such a way that the drug amine protonizes in the presence of the negatively charged phosphate oxygen of the phospholipid. Hexadecanoic acid is located deeper within the liposome surface than other negatively charged phospholipids. Correspondingly the drug action is weaker and drug protonization is prevented.

Keywords: electrophoretic light scattering; liposomes; drug binding; surface groups.

Introduction T h e r e c e p t o r h y p o t h e s i s of local a n e s t h e t i c action has b e e n put f o r w a r d by several investigators, w o r k i n g with q u a t e r n a r y derivatives of a n e s t h e t i c m o l e c u l e s . T h e s e c o m p o u n d s are s u p p o s e d n o t to p e n e t r a t e the n e r v e m e m b r a n e from o u t s i d e to inside a n d are f o u n d to be active o n l y w h e n a p p l i e d from the inside by i n t e r n a l p e r f u s i o n of the axon [1-3]. H o w e v e r the d e t e c t i o n or e v e n isolation of such an a n e s t h e t i c r e c e p t o r p r o t e i n has n o t b e e n successful u n t i l now. M o r e s u p p o r t has b e e n given to the h y p o t h e s i s of the lipid phase b e i n g the r e c e p t o r site for local a n e s t h e t i c action, b e c a u s e t h e r e is a strong c o r r e l a t i o n b e t w e e n a n e s t h e t i c p o t e n c y a n d the solubility of 0009-3084/83/$03.00 (~) 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

82 the molecule in a hydrocarbon environment [4,5]. Furthermore the wide variety of molecules possessing anesthetic activity led to the conclusion that the site of action is not a specific protein but rather the lipid core of the biological membrane which allows different non-specific interactions. Phospholipids are easily amenable for various studies. Phospholipid vesicles and black lipid bilayer membranes serve as good model systems for biological membranes. On such model membranes local anesthetics exert different effects: (1) the fluidity of the phospholipid molecules is increased [6--8]; (2) the membrane is expanded [4,9,10]; (3) the surface potential is changed [11-141. I n our recent study [14] we could demonstrate that drugs with local anesthetic properties increase the surface potential of electrostatically neutral hposomes from phosphatidylcholine and decrease that of negatively charged liposomes. Most of the experiments were performed with liposomes made from one single phospholipid. The biological membrane however is composed of ~tvariety of phospholipids with phosphatidylcholine and phosphgtidyletharlolamine being the components with the highest concentration [15]. In this study we mixed phosphatidylcholine as the major phospholipid wLth different classes of neutral and negatively charged phospholipids and with negatively and positively charged single chain lipids. The interaction of drugs with such liposomes having different surface chemical properties might be helpful to clarify the molecular mechanism of anesthetic action.

Materials and methods

The following phospho- and sphingolipids were used (all purchased as high purity lipids from Serdary Research Laboratories, London, Ontario, Canada N6G 2R7: lysophosphatidylcholine (LPC, pig liver), phosphatidylethanolamine (PE, pig liver), phosphatidylserine (PS, pig brain), phosphatidylinositoi (PI, pig liver), cardiolipin (CL, beef heart), phosphatidic acid (PA, from egg lecithin), sphingomyelin (SM, pig brain), cerebrosides (Cer, beef brain). Phosphatidylcholine (PC) was prepared from egg yolk and purified by column chromatography. The concentration was determined by analyzing the lipid phosphorus according to a slightly modified method of Gee and Deitz [16]. Hexadecanoic acid, hexadecylamine and hexadecyltrimethylammonium were purchased from Fluka, Buchs, S.G. Switzerland, as analytical reagents. Liposomes were prepared by mixing pure phosphatidylcholine dissolved in chloroform with appropriate volumes of one of the above mentioned lipids, dissolved in chloroform as well. The organic solvent was evaporated under vacuum and an appropriate buffer solution (1 mM KCI, 1 mM glycine, 0.3 mM EDTA, pH 7) was added under nitrogen atmosphere. The suspension was hand shaken for at least 5 min before it was sonicated in a probe-type sonicator (Branson sonifier B-15, 3-mm tip

83 diameter) at a nominal frequency of 20 kHz and lowest power level. The sonication times for the different probes varied, depending on the phospholipid used. The suspensions were exposed to ultrasonic irradiation, until they became opalescent (about 15 min for electrostatic neutral phospholipids and 30 s for negative phospholipids). The total lipid concentration was 0.3 mg/ml buffer in all experiments. The liposome mobility was determined by laser Doppler electrophoresis, as described previously [14]. From the mobility values the zeta potential and the total charge was calculated according to Gouy-Chapman theory. Converting the mobility values to zeta potentials, a value for Henry's function f ( K a ) = 1.32 was introduced into the equations [14,17]. This is based on a mean particle radius of a = 1700 ~ and Ka = 25. The size of the liposomes was determined from laser light scattering experiments. The width of the autocorrelation function in the absence of an electric field is a measure for the diffusion coefficient, which is related to the size of the particle by the Einstein-Stokes equation. A certain percentage of multilamellar liposomes cannot/be excluded in these preparations. But this is not important for t h e drug effects as in electrophoresis only net charges of particles contribute to the driving force. According to theory non-linearities b e t w e e n / z and ~" occur for Ka < 20 and ~ > 100 [18,19]. In our case Ka and ~" are very near where non-linearities are expected but comparative studies of drug effects on differently composed liposomes should be allowed [20]. The mobility values are means of at least two experiments with a deviation of < 5 % at low drug concentrations and of <2.5% at high drug concentrations. Lipid/buffer partition coefficients (P) were determined in the following way: handshaken liposomes from PC, PC plus 10 mol% PS and PC plus 10 moi% hexadecyltrimethylammonium were prepared in the same buffer used for the electrophoretic measurements. The drugs were added to 5 ml samples at three different concentrations. After centrifugation of the samples at 55 000 rev./min (Beckman, Ti SW 55) for 2 h the pellets were dissolved in ethanol. Drug concentrations in the supernatants and ethanol solutions were d e t e r m i n e d spectrophotometrically (293 nm propranolol, 312 nm tetracaine, 299 nm procaine). Partition coefficients were calculated from the ratio of anesthetic concentration in the lipid to that in the supernatant: P = Cl/Cs. No partition coefficients were determined for lidocaine because of the weak absorption maximum.

Results

The influence of drugs with different local anesthetic activity on the electrophoretic mobility of liposomes composed of. different lipids has been measured. The results are summarized in Fig. l a d . The liposomes are prepared from phosphatidylcholine, mixed with 10mo1% of the lipids

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85 mentioned above. All the drugs decrease the electrophoretic mobility of liposomes which contain negatively charged lipids and increase the mobility of iiposomes which are electrostatically neutral. Positively charged drugs neutralize negative surface charges of PS to zero mobility and at higher concentrations the liposomes become positively charged till, at about 6 mM, no further increase in mobility could be observed. The effect of lidocaine and procaine on the electrophoretic mobility of negatively charged liposomes is weaker, compared to propranolol and tetracaine. Only at concentrations of 0.1-0.2 mM first mobility changes are observed. Zero mobility (complete neutralization of the negative liposome surface) is not achieved, even at 6 mM drug concentrations. The mobility-concentration plots in Fig. l a d show the same slopes for liposomes containing 10mol% of either PI, PS and PA, which are all negatively charged phospholipids. The control values without drugs demonstrate the different contributions of the three phospholipids to the surface charge. PA contribution is larger than PS and this larger than PI. Cardiolipin, structurally consisting of two PA molecules linked by a glycerol chain, shows the highest mobility values under control conditions. Liposomes containing hexadecanoic acid show different behavior. The control value of these liposomes is much smaller than that of other negatively charged lipids (Table I), although the same amount is mixed with PC (10mol%), and the pK of hexadecanoc acid and of phosphatidylserine carboxylate is about the same, 4.8. According to the reduced control value the drug effects are moderate as well. In case of hexadecylamine and hexadecyltrimethylammonium, liposomes are positively charged and the drug effect is blocked. No significant change in electrophoretic mobility even under the strong acting drugs like propranolol and tetracaine is observed. Liposomes prepared from PC and 10mol% of other electrostatically neutral or uncharged phospholipids like LPC, PE, SM and Cer showed almost the same effects under drug action as liposomes from pure PC alone. There were slight variations among these lipids, however not exceeding mobility values of ___0.15. It is not of great importance for the drug effect whether the liposomes contain single chain lipids (LPC), a phospholipid with an ethanolamine group instead of choline (PE), a phospholipid with different chain lengths (SM) or a lipid, where the choline and phosphate group are missing (Cer). Table I summarizes the results of this study. Besides the zeta potential values for the different liposome experiments under control conditions the potential changes due to a ten-fold increase in drug concentration are also listed. These potential changes are taken from the linear part of the effect-concentration curves, i.e. from 0.04 to 0.4 mM propranolol and tetracaine and from 0.6 to 6 mM lidocaine and procaine.

86 TABLE I C H A N G E S IN Z E T A P O T E N T I A L BY P R O P R A N O L O L , T E T R A C A I N E , L I D O C A I N E AND PROCAINE OF LIPOSOMES COMPOSED OF PHOSPHATIDYLCHOLINE AND 10 tool% O F T H E LIPIDS L I S T E D IN T H E F I R S T C O L U M N

Lipid

Changes in zeta potential (mV) Control

Cardiolipin Phosphatidic acid Phosphatidylserine Phosphatidylinositol Hexadecanoic acid Phosphatidylcholine Lysophosphatidylcholine Phosphatidylethanolamine Sphingomyelin Cerebrosides

Propranolol

Tetracaine

Lidocaine

Procaine

(0.04-0.4 raM)

(0.04-0.4 mM)

(0.6--6 raM)

(0.6--6 raM)

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86.8

62.7

38.9

39.7

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64.3

57.0

43.7

43.1

-98.0

66.7

57.5

37.8

39.0

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65.1

54.1

39.2

39.0

-27.8

33.6

23.6

14.9

9.6

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31.0

26.7

14.5

11.2

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29.4

28.6

14.1

12.2

-17.7 -8.0 -8.8

32.9 27.0 28.1

31.8 24.1 25.9

15.4 10.4 13.2

9.8 8.2 10.3

The values show that negative surface charges facilitate drug binding, with propranolol being the most effective drug, followed by tetracaine, lidocaine and procaine. The effects are smaller in electrostatically neutral liposomes. Liposomes formed from PC and neutral lipids show small negative surface potentials under control conditions. These cannot be explained according to the chemical formula and may be due to small impurities in the different lipid preparations. The influence of surface charge on the drug effect is investigated in more detail in a second set of experiments, where the concentration of phosphatidylserine and of hexadecanoic acid is successively changed. The results are shown in Fig. 2a-d. A similar dependence of liposome mobility, i.e. zeta potential on drug concentration is observed as in Fig. l a d . A higher concentration of negative charge in the liposome surface either by hexadecanoic acid or by phosphatidylserine increase drug binding. The drug effect on hexadecanoic acid containing liposomes is not as strong as on phosphatidylserine liposomes. This becomes most obvious when the drug induced changes of mobility for liposomes containing 20 mol% hexadecanoic acid are compared with the ones of liposomes containing 3 mol% phosphatidylserine. These results are

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89 comparable because both liposome preparations have about the same control values. As derived from the slopes the interaction of the drugs with liposomes containing hexadecanoic acid is weaker than with phosphatidylserine liposomes. Table II summarizes data obtained from partition coefficient measurements of the drugs in three differently composed lipid samples and of the drug induced charge per lipid molecule, as calculated from the values in Fig. 1 by Gouy-Chapman equations. Propranolol shows the highest partition coefficient, followed by tetracaine and procaine. Coefficients strongly increase when the liposomes contain negatively charged PS and have about the same values as in PC when the liposomes contain positively charged hexadecyltrimethylammonium. With decreasing drug concentration a higher amount is found in the lipid phase in relation to the buffer medium. For convenience the drug concentrations in the lipid phase are recalculated as drug molecules per lipid molecules, where the drug is normalized to 1. While the partition coefficients increase with decreasing drug concentrations the drug/lipid ratios decrease. The charge per lipid is calculated from the drug induced zeta potential change (A~ from control to actual value), assuming an area of 6 5 ~ 2 per lipid molecule. The charge/lipid ratio parallels the drug/lipid ratio with values being generally smaller. When the liposomes contain PS, higher charge/lipid ratios are found than in PC alone. For 1 mM propranolol the charge/lipid ratio approximates that of drug/lipid indicating that almost all propranolol dissolved in the lipid phase is located at the liposome surface in its charged form. Very low charge/lipid ratios are measured in hexadecyltrimethylammonium containing liposomes, although the drug/lipid ratio is about the same as in pure PC. The positively charged lipid prevents the protonization of the drugs in the liposome surface.

Discussion

Laser light scattering experiments reported previously have shown that the liposome preparations in our studies are homogeneous in size and shape [14,21]. The drug induced changes in the surface potential of liposomes can clearly be related to a hydrophobic interaction of the drugs with the lipid phase in such a way that the polar protonated amine group points towards the hydrophilic lipid surface groups. This study gives several indications that the positively charged amine group of the drug is located near the negatively charged phosphate group of the phospholipid. The drugs exert about the same effects on PI, PA or PS containing liposomes as is derived from the control values and from the slopes of the effect concentration curves (Table I). PI and PA have a single negative charge from their phosphate oxygens, while PS contains in addition to the phosphate oxygen a negatively charged

90 carboxylic and positively charged amine group. PI and PS are phosphodiesters and therefore less charged than the phosphomonoester PA, which shows the highest mobility values. But also screening effects on the negative phosphate oxygen charge by bulky residues can partly be responsible for the reduced mobility values of PI. The identical slopes of the effect-concentration curves indicate that no structural related hindrance of drug binding by different phospholipid headgroups occurs and that the binding site is the negatively charged phosphate oxygen. When the PC liposomes are enriched with various electrostatically neutral lipids like PE, SM and Cer, about the same drug effect is observed. This indicates again that the interaction of the drugs is independent of the nature of the lipid head group, except it carries an extra positive or negative charge. Negative charges in the lipid surface facilitate drug binding. This is not only demonstrated by the results in Fig. 1 but also by the partition coefficients and drug/lipid ratios in Table II. Ten percent PS in PC causes a strong increase in partition coefficients, especially of those drugs which show a weak action on pure PC liposomes. For example the partition coefficient of 1 mM procaine in PC-PS is 5 times higher than in PC and that of 1 mM propranolol only twice as high. As can be seen from the drug/lipid and charge/lipid ratios, only a certain amount of the incorporated drug is in its protonated form at the surface of the liposome. PS increases the partition coefficient but not necessarily the amount of protonated drug. When the liposomes contain 10% positively charged hexadecyltrimethylammonium the partition coefficients are about the same as in PC but the amount of charged drug in the liposome surface is strongly reduced. The C16 single chain lipid is deeply incorporated into the lipid phase by Van der Waals forces with its positively charged amine group near the phosphate oxygen of the phospholipid. Occupying drug binding sites the drug is prevented from protonization and stays in its uncharged form within the lipid phase. The same interpretation holds for hexadecylamine, which, according to its pK value induces less positive charge than hexadecyltrimethylammonium. Hexadecanoic acid, a C16-single chain lipid with a terminal carboxylic group adds negative surface charge to PC liposomes. Although the same amount is used and the pK of its carboxylic group is about the same as for PS, the charge density is significantly less than in PI, PS or PA liposomes (Table II). This lipid probably incorporates deeper into the hydrophobic core of the lipid bilayer so that its charged group contributes less to the surface charge with the result of a weaker interaction of the drugs. An additional effect could arise from the influence of the saturated carbon chains of hexadecanoic acid on the thermodynamic properties of the surrounding phospholipid acyl chains. A shift of the phase transition temperatures of dimyristoyl- and dipalmitoylphosphatidylcholines by dodecanol or myristic

91 acid to higher temperatures has been measured by Lee [22,23]. This result can be interpreted in terms of a rigidifying effect of the long chain saturated hydrocarbons on the acyl chains of the surrounding phospholipids. A tighter packing of the phospholipid molecules prevents the incorporation of drugs. This also supports the hypothesis that the drugs are incorporated between the lipid molecules and are not bound to the liposome surface by ionic forces. That the drug molecules enter the hydrophobic phase of the lipid bilayer has been proved by several investigators, using various methods [4,6-9,24,25]. However the partition coefficient for PC plus 10% hexadecanoic acid proved to be 62% higher than for pure PC which shows that the incorporation of the drugs is facilitated and not prevented by the thermodynamic conditions of the lipid acyl chains. The slopes in Fig. 2a--d indicate that the drugs interact more strongly with liposomes which contain PS instead of hexadecanoic acid. Liposome preparations containing 3% PS or 20% hexadecanoic acid show the same mobility values under control conditions but different slopes in the effect-concentration relations. The slopes of the effect-concentration curves in Fig. la and b for PC and PC plus hexadecanoic acid are the same. The partition coefficient for hexadecanoic acid containing liposomes is higher than the one for PC which indicates that hexadecanoic acid prevents the drugs from protonization in the liposome surface. One could expect differences in drug/lipid partition coefficients when multilamellar or small bilayer vesicles are used. A significant difference however seems to be unlikely. The high values for the partition coefficients clearly indicate that most of the drug enters the lipid phase. Therefore, the influence of the vesicle structure on this process is negligible. Partition coefficients have also been reported for oil-, oleyl alcohol- and octanolbuffer systems where the organic phase, representing the lipid, is a homogenous solvent and not dispersed at all. The results demonstrate that the drugs enter the lipid phase in the uncharged form by pure hydrophobic interactions and then reprotonize in the surface, changing the surface potential. The reprotonization process needs negative phosphate groups. Similar mechanism could play an important role for the action of local anesthetics on nerves [1-3,12,26].

Acknowledgments The authors would like to thank Dr. L. Michaelis for critically reading the manuscript and Ms. S. Schimmelpfennig for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 30, Kardiologie.

92

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