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


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


Theory: The use of FPE to monitor membrane-protein interactions.

The present study makes use of the fluorescent probe, fluorescein-phosphatidylethanol-amine (FPE) which can be securely incorporated into liposome and cell membranes for reporting changes in the membrane surface potential, in response to protein-membrane interactions. The principles of this strategy are descibed by (34, 35, 36). A related technique described by Bergers et al (37) measured changes in the zeta potential associated with a pH titration of globular proteins bound to liposomes. This latter technique, however, is in principle less accurate than those obtained with FPE (34, 38, 39, 40).

Preparation of FPE labeled liposomes.

Negatively charged large unilamellar vesicles (LUVs) (41), with a diameter of 100nm were prepared using 85 mole% Palmitoyl-oleoyl-phosphatidylcholine (PC): 14 mole% Palmitoyl-oleoyl-phosphatidylserine (PS) : 0.5 mole % FPE. The method of preparation is essentially as described by Hope et al (42). Briefly, the phospholipid (stored in methanol: chloroform, 1:1 v/v) and the FPE (stored in methanol: chloroform, 1:5 v/v) were mixed in a round bottomed flask and dried under a stream of oxygen free, argon gas by rotary evaporation until a thin film was formed. This was best achieved by repeating the drying process after resolubilising the lipid mixture in pure chloroform. The lipid film was rehydrated using 1ml of the appropriate buffered solution and left to stand in the dark. The resulting multilamellar vesicle suspension was then freeze-thawed five times in liquid nitrogen (43) followed by aspiration, ten times, with a Pasteur pipette before being transferred to a device (Sciema Technical Services Ltd., Richmond, BC) for extrusion through standard 25mm diameter polycarbonate filters with a 100nm pore size (Nucleopore Filtration Products, CA, USA). The vesicles were injected into a chamber above the filter and nitrogen pressure applied from a gas cylinder fitted with a pressure regulator. The vesicles were extruded ten times under positive pressures of 250-550 lb/in2. The resulting unilamellar vesicles, made up a homogenous population with 90% having a diameter of 100nm and another 10% of 100 nm ± 10nm. The sample was collected and stored under argon, in the dark at 4°C until required for the experiments.

Vesicles were prepared and suspended in one of the following solutions: 280mM sucrose; 100mM potassium chloride; and 100mM potassium thiocyanate. All solutions contained 5mM Tris-base as a buffer with the pH set at 7.5 using HCl/KOH, at 20°C.

Culturing and FPE labelling of HOM-2 lymphocytes.

The Epstein-Barr virus transformed human lymphoblastoid cell line, HOM-2 (HLA phenotype A3, B7, DR1, DQw1, DPw4), was cultured in RPMI 1640 (Life Biotechnologies, USA) supplemented with 10% foetal calf serum. The cells were harvested and washed by two centrifugation steps at 2500xg for 5-10 seconds in PBS (150mM NaCl, 5mM Na2HPO4, pH 7.5). The cell number was determined by hemocytometer counting of a minimum of 100 cells. Cell viability was determined, by trypan blue exclusion (Sigma Chemical Co, St Louis, USA, application note). Cells were labelled with FPE according to the ratio, 10µg FPE : 3 x 106 cells. FPE was prepared for addition by removal of the chloroform-methanol solvent under a stream of argon gas followed by re-suspension in 15µl of 95% ethanol, to which the cell suspension was added in a maximum volume of 3ml. The mixture was gently agitated before incubation in the dark for 1 hour at 37°C. Unincorporated FPE was removed by three serial centrifugations at 2500xg for 10 seconds in the appropriate iso-osmotic buffered solution. The cells were stored in the dark at 4°C and used within 48 hours of preparation.

Agarose Gel Electrophoresis.

An estimation of the net charge of lambdaRG57, as well as the purity of protein samples were estimated by agarose gel electrophoresis. Electrophoresis was performed using either pre-cast, eight well, 1% agarose gels, prepared using sodium barbitone, pH 8.6 (Ciba Corning Diagnostics, Switzerland). Alternatively, 1% agarose gels at pH 7.5 and 9.5, in the same buffer, were cast in our laboratory. Samples were loaded onto the gel using plastic applicator tubes (Ciba Corning) which allowed a standard volume of 4µl to be accurately applied. Electrophoresis was performed using barbital buffer (50mM sodium barbital, 1mM EDTA, at pH 8.6) as the electrolyte, for 35-40 minutes at a constant voltage of 90mV. The gels were stained by immersion in a 0.2% w/v Amido black solution for approximately 10 minutes. Then, the gels were air dried overnight prior to destaining with a 5% v/v acetic acid solution.

Multiple Myeloma patients.

Urine from a patient with multiple myeloma was obtained with an informed consent from Severalls Hospital, Colchester UK, and urine was kept frozen at -70°C until used. The patient presented with a high level of a monoclonal paraprotein in the serum and urine. The paraprotein was a Bence Jones protein of lambda isotype and presented predominantly as a dimer in the urine, which was designated lambdaRG57. The amount of, lambdaRG57 in the urine was 6mg/ml lambda light chain. The amount of urine creatinine and serum levels of other immunoglobulins were normal.

Isolation of the myeloma paraprotein.

The myeloma paraprotein was isolated according to well established methods (44, 45), involving a concentration step followed by chromatographic purification.

The protein was concentrated in one of two ways:-

1) Ammonium sulphate precipitation, by adding ammonium sulphate to a final concentration of 50% (w/v), followed by incubation at 4°C whilst continuously stirring for 48 hours. The precipitate so formed was isolated by centrifugation at 10 000xg for 20 mins. The precipitate was re-dissolved in a small volume of 5PB at pH 7.5 and stored in 5ml aliquots at -70°C until used.

2) Size exclusion concentration employing, an AmiconTM concentrator and filter (Diaflow, UK) with a cut-off size for proteins greater than 10kD. The urine was forced through the filter under pressure using nitrogen gas at 200lb/in2. This was carried out in the dark at 4°C. The filtration continued until a twenty fold concentration was achieved. The resulting turbid protein solution was dissolved in a small volume of PB at pH 7.5 and stored in 5ml aliquots at -70°C until used.

The protein was isolated using G-75 (Sigma) size exclusion chromatography, and further purified by DE52 (Whatman, UK) anion exchange chromatography. After each procedure the purity of protein was monitored with agarose gel electrophoresis.

The protein concentration was monitored after each step essentially as described (46). Determinations of the absorbances of the protein solution at 235nm and 280nm against a suitable blank, were made, and the following equation employed:

Protein conc. (mg/ml)=Abs235nm - Abs280nm

Spectroscopic Analysis.

Circular dichroic spectra were obtained in PBS (pH 7.5) and sucrose solution (pH 7.5), in the presence and absence of FPE-free PC/PS phospholipid vesicles (PLVs), as indicated. In all cases, lambdaRG57 was at 2.17µM and the PLV sample contained 128µM lipid. This provided a protein: lipid ratio of 1 : 59 on a molar basis. Circular dichroic measurements were performed at room temperature, taking measurements at UV wavelengths, between 200nm to 260nm. Samples were analysed in a silica quartz cell with a 0.5mm pathlength on a Jasco J600 spectropolarimeter .

Fluorescence measurements were performed using a Perkin Elmer LS-50 spectrofluorimeter (Perkin Elmer, UK) with the excitation and emission wavelengths set at 490nm and 516nm respectively. Stopped-flow rapid mixing experiments were performed using an Applied Photophysics system (Leatherhead, UK), employing a 500nm cut-off filter, with the excitation wavelength set at 490nm. The time resolution of this apparatus was to within a few milliseconds. The data were imported into a data analysis package (Ultrafit, BIOSOFT, Ferguson, MO, USA) and the rate constants determined by fitting the curves to either a single or double exponential decay with offset, employing the following equations,

single exponential with offset: y=y0 · exp -k1t + c

double exponential with offset: y=y01 · exp -k1t + y02 · exp -k2t + c

Where y, is the intensity of the fluorescence signal, y01 and y02 are the initial fluorescence signals determined at t = 0, for rates k1 and k2 respectively, t is the time and c the value of the offset, which represents the endpoint of the exponential decay at t = infinity. The fitting used 95% confidence limits and was carried out using the Gauss-Newton-Marquardt algorithm with robust weighting which rejected outliers with a rejection factor of 6. The corresponding residuals were calculated and the "goodness of fit" was expressed as a X2 value.

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