Simple flow-cell for the fluorimetric study of conformational changes of proteins on agarose matrix

ANALYTICAL

BIOCHEMISTRY

60, 505511

(1974)

Simple Flow-Cell for the Fluorimetric Study of Conformational Changes of Proteins on Agarose Matrix ANDRIZ Laboratorium

0. BAREL

VOOT Algemene en Biologische Scheikunde, Fakulteit en Farmacie, H.I.L.O., Vrije Universiteit Brussel, Belgi6

Geneeskunde

AND

HENRI Laboratorium

voor

Biochemie,

ROOSENS

Fakulteit

Wetenschappen,

Vrije

Univewiteit

Brussel, Belgil Received October 24, 1973; accepted January 30, 1974 The construction and utilization of a new cylindrical cell for fluorescence measurements on protein-Sepharose 4B conjugates is described. This experimental device proved very convenient for fluorimetry of proteins covalently bound to agarose gels, for measurements on proteins in solution, and finally for monitoring the adsorption of proteins in the course of affinity chromatography. With the aid of this cell, the fluorescence spectra of human .cw-lactalbumin in solution and in an insoluble state were compared. The cY-lactalbumin-Sepharose 4B complex gives a spectrum which closely resembles that of the native protein. Fluorescence spectra were recorded with as little as 50 pliters gel in the cell, which corresponds to approximately 0.015-23 nmoles of chemically bound protein. The fluorescence intensity was within experimental error proportional to protein concentration from 0.03 to 0.20 nmole bound protein/mg dry resin. The application to conformational studies on membrane bound of the lactose synthetase function is discussed.

of this enzymes

fluorimetric method such as the proteins

INTRODUCTION Fluorescence has been used extensively these last years as a tool for of conformational changes in proteins. In addition, enzymes and proteins bound to insoluble carriers present a fundamental interest as model systems for membrane bound proteins and affinity chromatography. A fluorimetric study on insolubilized chymotrypsin and trypsin, using a rectangular front face cell, was recently described by Gabel et al. (1).

the study

Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

505 Inc. reserved.

506

BAREL

AND

ROOSENS

However, according to the description given by these authors, the use of this fluorescence cell for routine measurements seems somewhat difficult since the assembling of the cell parts requires gluing and precise adjustment. In the present paper we describe the construction and the performance of a very simple cylindrical cell which can be used for fluorescence measurements on proteins covalently bound to Sepharose gels. Besides, the potential use of this device as flow cell for column affinity chromatography with proteins on insoluble agarose gels is presented. MATERIALS

AND

METHODS

Materials

Crude human a-lactalbumin was prepared by the method described by Aschaffenburg (2) for bovine milk. Further purification was achieved by gel filtration on Sephadex G-100 (3) and chromatography on DEAEcellulose (4). Sepharose 4BYa-lactalbumin complex was prepared according to the method described by Trayer and Hill (5). The protein content of the complex in the fluorescence cell was estimated by amino acid analysis. The total amount of gel was quantitatively transferred into a Pyrex hydrolysis tube. The sample was hydrolyzed in 6 N hydrochloric acid for 24 hr at 105°C and thereafter evaporated to dryness. The dry residue was suspended in 0.01 M pyridine-acetic acid buffer and passed through a small column of Dowex 50W X 2 (H+ form) in order to eliminate the black charred sugars of the Sepharose mat,rix. The eluate was dried and subjected to amino acid analysis according to the method of Spackman et al. (6). Knowing the amino acid composition of human ly-lactalbumin (3) it was possible to estimate the protein content of the fluorescence cell with reasonable accuracy ( + 10%). Construction of the Cell

The basic design is illustrated by the diagram in Fig. 1. The cell consists of a stainless steel top end piece (A) with Teflon tubing and a fitting, Beckman part no. 313 336, (B), a Teflon adapter (C), fitting into a cylindrical microcell (D) , and a stainless steel bottom end piece (E) , with Teflon tubing and a stainless steel screw adapter (F). The microcell (quartz, 5.0 mm o.d. and 3.0 mm i.d.) was obtained from American Instrument Co., cat. no. A3-64051. The bottom of the cell was cut off with a diamond disk and replaced by a tight fitting porous Teflon disk (G). An appropriate holder (H) for this cell was made of aluminium and fits into the ordinary cell compartment of the fluorimeter. It was necessary to remove

FLUORESCENCE

OF

PROTEIN-SEPHAROSE

507

CONJUGATES

4 1I I

A

--I; II I I I

1

--

X

I.; !-g

I I

-: , 0 .-

f I I

I I I I

57 --X’

I I

I I I

In f-i

I

FIG. 1. Diagram in millimeters.

of the fluorescence cell. Except otherwise

indicated

I

all lengths are

the existing screw button fixing the cell compartment to the apparatus; it was replaced by a bored screw in order to pass the Teflon outlet tubing. A square microflow cell (such as the flow cell supplied by American Instrument Co. cat. no. 4-8114) can also be used as fluorescence cell for measurements on proteins in solution and in an insoluble state (1). There are, however, several advantages to using the cylindrical cell even though scatter may be somewhat great,er than for rectangular cells. First, construction is very simple. Second, the ends of a cylindrical cell are easy to

508

BAREL

AND

ROOSENS

seal with O-rings for its use as a chromatographic column. Third, it can be easily cleaned for reuse and fourth, the amount of material required for the cylindrical system is half that required by a commercially available rectangular cell (i.e., 50 pliters vs 100 pliters). Fluorimeter

An Aminco Bowman SPF spectrofluorimeter was used in this study. The apparatus is equipped with a RCA R4465 phototube, an off axis ellipsoidal condensing system, and a Houston 2000 X-Y recorder. Correction of the emission spectra was found unnecessary since the spectral response of the detection system (i.e., monochromator plus photomultiplier) remains reasonably constant (? 10%) from 300 t.o 500 nm. It was necessary to narrow the exciting beam with slits of 1 mm in order to diminish the scattering off the round cell. With the combination of slits used, the excitation and emission bandwidths were 10 nm. All experiments were carried out at room temperature. Fluorescence

Measurements

In order to obtain reproducible fluorescence spectra on insoluble proteins it is necessary to follow the following experimental procedure. Starting from the completely deassembled cell, the microcell (D and G) (see Fig. 1) was filled with maximum 100 Jiters (usually 50 pliters is enough) of the protein agarose gel by means of a Pasteur pipette. The gel was immediately layered with 100 pliters of the appropriate eluting buffer. The Teflon adapter (C) was fitted into the microcell and the cell partially assembled (C, D, G, E, and F). Finally, the cell was introduced into the cell holder (H) and secured in place with the top end piece (A). The eluting buffer was then passed downward through the gel with the aid of a Vario Perpex LKB peristaltic pump. A flow rate varying from 0.2 ml to 0.5 ml/min was used. All the spectra were recorded after the gel had been washed during 10-15 min with the buffer. The protein-Sepharose 4B gels were illuminated only during the time necessary to record the emission spectra, in order to minimize photoinactivation. By following this procedure, the protein-gel aliquot could be repeatedly used and it was shown that photoinactivation of the protein sample was negligible. We were able in this study to record easily the fluorescence spectra of about 0.2300 yg of protein (in this case cy-lactalbumin) covalently linked to Sepharose 4B. It is worthy to note that the same cell device can be used for classical fluorimetry on protein samples in solution. In this case the protein solution was introduced upward into the flow cell. This procedure allowed us, while maintaining the overall cell geometry constant, to compare the fluorescence spectra of proteins in solution and on an insoluble matrix.

FLUORESCENCE

OF PR0TEIN-SEPHAROSE

CONJCGATFS

The a-lactalbumin solutions were usually studied at a concentration ing from 0.05 to 0.15 mg/ml in 0.05 M Mops buffer at pH 7.5. RESULTS

AND

509 rang-

DISCUSSION

The fluorimetric cell described in Materials and Methods shows some improvements over the fluorescence cell of Gabel et al. (1). Among these improvements we would like to point out: (a) The ease of assembly and disassembly of the cell for routine measurements, (b) no glue is necessary in order to ensure water tightness and, more importantly, (c) the versatility of the cell which can be used as an ordinary cylindrical cell, as a flow cell or as a micro column for affinity chromatography. In order to illustrate some of t,he above mentioned features, the fluorescence spectra of human a-Iactalbumin free in solution, of human cu-lactalbumin-Sepharose 4B complex and of Sepharose 4B alone are presented in Fig. 2. Sepharose 4B did not show any response to the excitation between 300 and 500 nm, excepting the normal scattering peak. The Iy-Iactalbumin-Sepharose 4B complex showed a maximum around 334-335 nm when excited at 280 nm. The peak of scattered light was reasonably well separated from the emission peak. The spectrum of human a-lact,albumin in solution was also recorded using the same cell assembly, and was found very similar to the spectrum of the agarose bound protein. So displace-

Wavelengfh

(nm)

FIG. 2. Fluorescence spectrum of human a-lactalbumin in solution (---), of Sepharose 4B alone (-.-.I and of human cu-lactalbumin-Sepharose 4B complex (---I. All spectra were performed in 0.05 M Mops buffer at pH 7.5 and at ordinary temperature. Excitation wavelength was 280 nm.

510

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ment of the maximum of emission could be observed between both spectra. Essentially similar results were obtained by Gabel and coworkers (1) on insolubilized trypsin and chymotrypsin. As already noted in the experimental part of this work, only very small amounts of covalently bound protein are necessary for each measurement. The concentrations of cu-lactalbumin fixed to Sepharose 4B used in this study varied from 0.03 to 9.0 nmole of protein/mg dry wt resin (this amounts to 0.3450 nmole protein/ml agarose gel). Since it was possible to record the fluorescence spectra with good accuracy with about 50 pliters of protein gel slurry this corresponds to 0.915-23 nmoles material. The fluorescence intensity of the a-lactalbumin-Sepharose 4B conjugate was investigated in function of bound protein concentration. As shown in Fig. 3, the fluorescence intensity was within experimental error proportional to protein concentration in the range of concentration from 0.03 to 0.20 nmole bound protein/mg dry resin. At concentrations of bound 1ylactalbumin higher than 0.25 nmole/mg dry resin significantly larger deviation to proportionality was observed. Therefore reproducible fluorescence spectra were recorded routinely in the range of concentration from 0.03 to 0.25 nmole of chemically bound protein/mg dry resin. Furthermore the position of the maximum remained constant within experimental error (l-2 nm). Finally we would like to point out that this experimental device can also be used as a microcolumn. Therefore the effect of various physicochemical parameters such as temperature, pH, denaturating agents, in-

Concentration

of bound

protein

nmole

per m g dry resin

FIQ. 3. Fluorescence intensity of cu-lactalbumin-Sepharose 4B conjugate at 334335 nm in function of bound protein concentration. All spectra were performed in 0.05~ Mops buffer at pH 7.5 and at ordinary temperature. Excitation wavelength was 280 nm.

FLUORESCENCE

OF PROTEIN-SEPHAROSE

CONJUGATES

511

hibitors, and effecters on the conformation of a covalently bound protein can be easily and rapidly investigated simply by changing the nature of the eluant flowing through the fluorescence cell. We are presently studying the effect of various factors on the two proteins (a-lactalbumin and protein A) of the lactose synthetase function (3,7-10). ACKNOWLEDGMENTS This work was supported by Grants from the National Fends voor Wetenschappelijk Onderzoek and the Nationale Raad voor Wetenechapsbeleid. We gratefully acknowledge Professor E. Schram for his interest in this work and helpful discussions. The authors thank Mr. J. P. Prieels for assistance in the preparation of the cr-lactalbumin-Sepharose 4B conjugates. REFERENCES I. Z., AND KATCHALSKI, E. (1971) Biochemistry 10, 4661. R. (1968) J. Dairy Sci. 51, 1295. B. C., AND BREW, K. (1972) Eur. J. B&hem. 27,65. 4. BARMAN, T. E. (1970) B&him. Biophys. Acta 214,242. 5. TRAYER, I. P., AND HILL, R. L. (1971) J. Biol. Chem. 246, 6666. 6. SPACKMAN, D. H., STEIN, W. H.) AND MOORE, S. (1968) J. Anal. Chem. 30, 1190. 7. RAMSWARUP, M., MORISSON, J. F.: AND EBNER (1971) J. Viol. Chem. 246, 7106. 8. KLEE, W. A., AND KLEE, C. B. (1972) J. Biol. Chem. 247, 2336. 9. BARKER, R., OLSEN, K. W., SHAPER, J. H., AND HILL, R. L. (1972) J. Biol. Chem. 1. GABEL,

D.,

STEINBERG,

2. ASCHAFFENBURG, 3. FINDLAY, J.

247,

10.

IVATT,

7135.

R. J.,

AND

ROSEMEYER,

M. A. (1972) FEBS Lett. 28, 195.