[Frontiers in Bioscience 1, a46-58, August 16, 1996]
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CAVEAT LECTOR



A STUDY OF THE INTERACTIONS OF AN IMMUNOGLOBULIN LIGHT CHAIN WITH ARTIFICIAL AND B-LYMPHOCYTE MEMBRANES.

Jonathan S. Wall1, Fayad M. Ayoub2, and Paul S. O'Shea

1 Human Immunology & Cancer Program, University of Tennessee Medical Center at Knoxville, 1924 Alcoa Highway, Knoxville, TN 37920-6999. USA.

2 Department of Biological & Chemical Sciences, University of Essex, Colchester, Essex, England U.K.

Received 01/11/96; Accepted 05/29/96; On-line 08/16/96

RESULTS

A qualitative measurement of the net charge of lambdaRG57 was determined by agarose gel electrophoresis performed at pH values of 8.6, 7.5 and 9.5 (Fig. 1). At pH 8.6, the protein demonstrated slight electrophoretic mobility towards the cathode, suggesting that its net charge at this pH is positive, implying a pK ~8.8. The protein therefore possesses a net positive charge at pH 7.5, and a net negative charge at pH 9.5 (Fig. 1).

Fig. 1 Agarose gel electrophoresis of lambdaRG57. The samples were placed on a 1% agarose gel equilibrated to pH 7.5 (lanes 1 and 2), pH 8.6 (lane 3) or pH 9.5 (lane 4), and subjected to a constant voltage of 90V for 35 minutes. Lanes 2, 3 and 4 contain 10µg of purified lambdaRG57. The position of BSA (20µg/well) at pH 7.5, is shown for comparison.

The interaction of lambdaRG57 with PC and PC/PS PLVs, at pH 7.5, generally resulted in a decrease in the steady state fluorescence intensity of the membrane bound FPE (Fig. 2).

Fig. 2 The interaction of llambdaRG57 with PC and PC/PS PLVs.

PLVs were suspended at a concentration of 80M lipid, in either, 280mM sucrose (A), 100mM KSCN (B), 150mM NaCl (C) or 100mM KCl (D), all supplemented with 5mM Tris at pH 7.5 and 20°C. Single additions of lambdaRG57 were made to the cuvette, after approximately 30 seconds, to give a final concentration of 39µM. The emission at 518nm was measured whilst exciting at 490nm.

The exceptions to this rule were found when PLVs were suspended in a medium containing 100mM KSCN. The changes in the fluorescence intensity of FPE, associated with the addition of lambdaRG57 to PC and PC/PS PLVs, are summarised in table 1.

Table 1. Summary of the mean changes in the fluorescence intensity of FPE incorporated into PC and PC/PS PLVs, upon the addition of lambdaRG57.

PLV compositionSuspending medium
(containing 5mM Tris at pH7.5)
Mean % change in
fluorescence intensity ± SE

PC100mM KCl-4.3 ± 0.8
PC/PS100mM KCl-8.0 ± 2.4
PC100mM KSCN< 1
PC/PS100mM KSCN< 1
PC280mM sucrose-7.3 ± 1.6
PC/PS280mM sucrose-8.8 ± 2.6
PC150mM NaCl-8.2 ± 2.3
PC/PS150mM NaCl-6.2 ± 0.7

The presence of 100mM KSCN in the suspending medium completely abrogates any change in the fluorescence intensity upon the addition of lambdaRG57 to either PC or PC/PS PLVs (Fig. 2A & Table 1). In contrast, the fluorescence intensity of FPE labelled PLVs suspended in 100mM KCl, was found to decrease upon the addition of lambdaRG57 (Fig. 2D & Table 1). The interaction of lambdaRG57 with PLVs suspended in either 150mM NaCl or 280mM sucrose solutions, resulted in a similar decrease in the fluorescence intensity. The change in the fluorescence intensity, in these cases, appeared to be independent of the charge carried by the PLV, as demonstrated by the parity of the signals for PC and PC/PS PLVs (Fig. 2A and C).

A decrease in the fluorescence intensity of the membrane bound FPE, suggests that Psis is becoming more electronegative due to the association of lambdaRG57 with the membrane surface. This observation appears to disagree with the prediction of the net charge of the protein shown by its electrophoretic mobility on an agarose gel (Fig 1). Considering the apparently conflicting fluorescence and electrophoretic mobility data, it is clear that the interaction of lambdaRG57 with the PLV membrane appears not to be a simple adsorption event. In other words, the apparent charge of the protein on the membrane surface differs from that in solution.

Finally, in an attempt to resolve in time, the events associated with the interaction of lambdaRG57 with charged PC/PS membranes system, a series of stopped flow rapid mixing experiments were undertaken. Mixing of the protein and liposomes results in a rapid increase in the fluorescence intensity (Fig. 3). A second phase which was slower and a decrease in the fluorescence intensity was also observed (with a half-time of ca. 20 seconds). An important feature of these data is the time over which these two events occur, the binding phase is rapid and is described by a single exponential decay with a calculated rate constant of k = 30.9 ± 1.2 sec-1. The secondary event, may also be described by a single exponential decay k = 0.031 ± 0.003 sec-1, but takes place over a period of time which is three orders of magnitude slower than the binding.

Fig. 3 Time resolution of the series of events associated with the interaction of lambdaRG57 with PC/PS vesicles. Rapid mixing experiments were performed by combining solutions containing 0.2mg/ml (4.34mM) lambdaRG57 and 0.2mg/ml (256µM) PC/PS vesicles in sucrose solution (280mM sucrose, 5mM Tris, pH 7.5) were prepared to allow for the dilution effect associated with the mixing process in the stopped flow apparatus. The excitation wavelength was set to 490nm and a 500nm cut-off filter allowed the acquisition of all light above this wavelength. Eight scans were taken and the average signal displayed, the data were analysed as described in Materials and Methods. A) The fast, binding phase, B) The slow reorganisation event. The residuals of the analysis are shown below the experimental data.

Identical binding experiments were performed at increasing temperatures up to 37.8°C (Fig. 4).

Fig. 4 Temperature dependence effects on the binding phase of lambdaRG57 with PC/PS vesicles. Additions of lambdaRG57 were made to PC/PS vesicles as described in figure 3, at the indicated temperatures.

The extent of binding, defined as the difference between the starting fluorescence intensity and that at equilibrium, decreased markedly as the temperature increased. This effect was accompanied by an increase in the rate constants (Table 3). Experiments performed at 2.8°C and 11.8°C (Fig. 5 and Fig. 6) once fitted to double exponentials made it clear that the second rate, associated with the binding process, remained constant, at 1.5 mol-1sec-1.

Fig. 5 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 2.8°C. Experiments were performed as described in figure 3, the temperature was set to 2.8°C. The double (solid line) and single (dotted line) exponential analyses of the data are shown together with the double exponential residuals (upper panel), the single exponential residuals (dotted line) are overlaid in the bottom panel.

Fig. 6 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 11.8°C. Experiments were performed as described in Figure 3, the temperature was set to 11.8°C. The double (solid line) and single (dotted line) exponential analyses of the data are shown. The double exponential residuals are given (upper panel) upon which the single exponential residuals (dotted line) have been overlaid in the bottom panel.

From this observation, k2 was deemed to be temperature insensitive and was fixed at 1.5 whilst the data taken at 24.8°C and 37.8°C were fitted to a double exponential decay. In order to determine whether the binding phase of lambdaRG57 to PC/PS vesicles was indicative of a single or double exponential process with a temperature insensitive second rate, both equations were used to fit the data and the residuals and X2 values for the goodness of fit were noted. The unweighted residuals for the data taken at 24.8°C were very similar when the data were fitted using either a single or double exponential (Fig. 7), and the X2 values were almost identical (Table 2).

Fig. 7 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 24.8°C. Experiments were performed as described in Figure 3, the temperature was set to 25.8°C. The double exponential analysis of the data is shown together with the corresponding residuals (upper panel), the single exponential residuals (dotted line) are overlaid in the bottom panel.

This suggests that the data can be adequately represented by a double exponential function. An investigation was undertaken as to whether the data collected at the two lowest temperatures could be fitted equally well, to a single exponential equally well, was undertaken (Fig. 5 and Fig. 6). It can be clearly seen from the residuals and fitted curves, that a fit to a single exponential was far less satisfactory than that to a double exponential function. This is borne out by a comparison of the X2 values for the residuals shown in Table 2.

Table 2. Statistical results for the "goodness of fit", the data represents the binding, fast phase, of the interaction of lambdaRG57 with PC/PS liposomes in a low ionic strength solution, at the indicated temperatures.
Temperature °CX2 for Double ExponentialX2 for Single Exponential
2.82.55E-44.86E-4
11.82.41E-44.93E-4
25.81.26E-41.71E-4

Table 3. The rate constants determined by stopped flow technique, for the interaction of lambdaRG57 with PC/PS PLVs. Experiments were performed as described for Fig. 3, at increasing temperatures. The k1 was fixed at 1.5 before fitting the data set.
k1- binding
(mol-1sec-1)
k2- binding
(mol-1sec-1)
k- secondary event
(sec-1)
2.81.520.77.1 x 10-4
11.81.526.01.8 x 10-3
24.81.546.03.1 x 10-2
37.81.598.412.2

Fig. 4 Temperature dependence effects on the binding phase of lambdaRG57 with PC/PS vesicles. Additions of lambdaRG57 were made to PC/PS vesicles as described in figure 3, at the indicated temperatures.

Fig. 5 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 2.8°C. Experiments were performed as described in figure 3, the temperature was set to 2.8°C. The double (solid line) and single (dotted line) exponential analyses of the data are shown together with the double exponential residuals (upper panel), the single exponential residuals (dotted line) are overlaid in the bottom panel.

Fig. 6 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 11.8°C. Experiments were performed as described in Figure 3, the temperature was set to 11.8°C. The double (solid line) and single (dotted line) exponential analyses of the data are shown. The double exponential residuals are given (upper panel) upon which the single exponential residuals (dotted line) have been overlaid in the bottom panel.

Fig. 7 Comparison of the single and double exponential analysis for the binding phase of lambdaRG57 to PC/PS vesicle at 24.8°C. Experiments were performed as described in Figure 3, the temperature was set to 25.8°C. The double exponential analysis of the data is shown together with the corresponding residuals (upper panel), the single exponential residuals (dotted line) are overlaid in the bottom panel.

The calculated rate constants for the double exponential analysis of the interaction of lambdaRG57 with PC/PS PLVs, at all temperatures are summarised in Table 3. Also included are the calculated rate constants for the secondary, slower event, at each temperature. The secondary event is adequately described by a single exponential decay at all the experimental temperatures studied, with a mean X2 value of 1.4 ± 0.4 x 10-4 for all four temperatures studied.

The data in Table 3 were used to construct an Arrhenius plot (see e.g., 47) using the following equation,

k=A exp (-Ea/RT)

where, k is the rate constant, A and Ea are the pre-exponential factor and the activation energy (KJmol-1) respectively, R and T have their usual meanings. For relatively simple processes, a plot of (ln k) against 1/T is anticipated to be linear, the gradient of which yields the activation energy. The results of an Arrhenius plot of lambdaRG57 binding to PC/PS vesicles in a medium of low ionic strength are shown in Figure 10. The temperature sensitive rate associated with binding was fitted by linear regression with an R2 value of 0.93 (1 is a perfect fit). Accordingly, the calculated energy for the temperature sensitive process was 13.53 KJmol-1. The secondary event yielded an activation energy of 87.89 KJmol-1 with an R2 value of 0.98 (Fig. 8).

Fig. 8 Arrhenius plot for the temperature sensitive rate of the binding of lambdaRG57 to PC/PS vesicles. Arrhenius plot of the binding of llambdaRG57 to PC/PS vesicles. Constructed using the rate constants shown in table 4. The data were fitted by linear regression with. The calculated value of Ea for the binding phase is 13.53 KJmol-1, and that for the slower reorganisation event is 87.79 Kjmol-1.

The presence of liposomes and a high and low ionic strength bulk phase on the secondary structure of lambdaRG57 was assessed by circular dichroic measurements. No change in the secondary structure was witnessed under these conditions. As shown in Fig 9, the protein remaining in its predominantly b-sheet configuration as indicated by the lack of change at the 218nm minima.

Fig. 9 Circular dichroic study of lambdaRG57 in high and low ionic media and in the presence and absence of PC/PS vesicles. Circular dichroic spectra were taken in PBS (150mM NaCl, 5mM Na2PO4) and sucrose solution (280mM sucrose, 5mM Tris, pH 7.5), in the presence and absence of PC/PS vesicles without FPE, as indicated. In all cases lambdaRG57 was at 0.1mg/ml (2.17mM), the liposome sample contained 0.1mg/ml (128M) lipid. This provided a protein to lipid ratio of 1: 58 on a molar basis.

An approximation of the isoelectric point of lambdaRG57 was determined by agarose gel electrophoresis at three different pH values (Fig. 1). At pH 8.6, lambdaRG57 carries a slight positive net charge as shown by its' electrophoretic movement to the cathode (Fig. 1). At the experimental and physio logical pH of 7.5, the protein is unequivocally cationic and similarly at pH 9.5 lambdaRG57 is anionic (Fig. 1). Although this is a crude representation of the pI of the protein, it provides sufficient information for the interpretation of their binding to membranes, as revealed by the changes in the fluorescence intensity of FPE.

The interaction of lambdaRG57 with FPE labelled PC and PC/PS PLVs in various suspending media generally resulted in a decrease in the fluorescence under the experimental conditions investigated (Fig. 2). PLVs suspended in 100mM KSCN however, exhibited no change in the fluorescence intensity upon the addition of lambdaRG57.

The role of the hydrophobic effect in the interaction of lambdaRG57 with PLV membrane surfaces has been investigated using the chaotrope KSCN (48). KSCN exhibits a high entropy of hydration and thereby, interferes with hydrophobic interactions (Fig. 4). The partial molar entropies of hydration of SCN- and Cl- have been quoted as -33.92 and -76.20 Jmol-1K-1, respectively (49). Binding was completely abrogated in 100mM KSCN, with respect to 100mM KCl implying that the hydrophobic effect, appears to play a major role in the binding process.

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