ANALYTICAL

BIOCHEMISTRY

87, l- 10 (1978)

A Sensitive Microassay for Protein Cells Cultured on Collagen

in

L. J. WALLACE AND L. M. PARTLOW Department

of Pharmacology, University of Utah, Salt Lake City, Utah 84132

College

of Medicine.

Received May 10, 1977; accepted January 6, 1978 A microassay for protein that is linear from 0.1 to 5 pg of protein and does not detect collagen has been developed. The assay is based on the ability of bromosulphophthalein (BSP) to form BSP-protein complexes which precipitate at low pH. Maximum precipitation occurs when 30 or more BSP molecules are bound per albumin molecule. Collagen is not detected because too little BSP binds to this protein to precipitate it. This assay should be of great value to those who grow dispersed cell cultures on a collagen substrate.

Dye-binding methods have been employed for the analysis of proteins because of their simplicity and sensitivity (1,2). Bromosulphophthalein (BSP) is one of the dyes of choice because of its great range of linearity (2). Even though BSP binds relatively nonselectively to most proteins, Oh ef al. (4), recently reported that the BSP protein assay developed by Nayyar and Glick (3) does not detect rat-tail collagen. Thus, this method should be especially valuable for measurement of protein in cells cultured on a collagen-coated surface (5-7). Unfortunately, the utility of existing BSP dye-binding methods is limited because of insufficient sensitivity (‘1 pg of protein; refs. 3,8,9) unless volumes are reduced and the optical density is determined by use of a microscope photometer and capillary cuvettes (3). Modifications to the method developed by Nayyar and Glick (3) which greatly increase the assay’s sensitivity without use of very small volumes are presented in this paper. In both the new microassay and the Nayyar and Glick (3) technique, maximum precipitation was shown to occur when 30 or more molecules of BSP are bound per albumin molecule; BSP/ protein molar ratios lower than 30 resulted in a reduction in the amount of precipitated BSP. In addition, collagen was not found to bind enough BSP to precipitate and was therefore not detected by the assay. METHODS

The protein assay developed by Nayyar and Glick (3) was carried out as described by Bonting and Jones (8) except for the following minor changes. 1

0003.2697/78/0871-0001$02.00/O Copynght 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

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First, the BSP was purchased from Gotham Pharmaceutical Co., New York (sulphobromophthalein sodium injection, USP). Second, polypropylene microcentrifuge tubes (A. H. Thomas, Philadelphia, Pa.) were used in place of glass test tubes. Third, centrifugation was carried out in a Beckman Model 152 Microfuge for 5 min at full speed. The amount of BSP bound to protein was determined by equilibrium dialysis. A large volume of a mixture of alkalinized protein and BSP in citrate buffer was prepared in the proportions given by Nayyar and Glick (3). Two milliliters of this solution was placed in a dialysis bag (12,000 MW cut off, A. H. Thomas) and dialyzed against 18 ml of an identical buffer solution lacking BSP and protein. Dialysis was carried out in glass scintillation vials (Packard Instrument Co., Downers Grove, Ill.) with constant stirring. At the times indicated in Fig. 2,45 ~1 was removed from the bathing solution and mixed with 65 ~1 of 4 N NaOH. The concentration of BSP in the dialysis bath was then quantified by measurement of the optical density of the alkalinized solution at 580 nm. The quantity of protein-bound BSP was determined at equilibrium by subtracting the total amount of BSP in the solution bathing a protein-containing sample from that in the control dialysis bath. The extent of aggregation of BSP-protein complexes was assessed by light scattering. Solutions containing varying amounts of protein were prepared as described for equilibrium dialysis. Light scattering was quantified at 5 10 nm using a Fart-and fluorometer (Valhalla, N. Y.) with two primary filters (Corning glass, number 3384 and 5543) and no secondary filters. The light-scattering data given in Fig. 3 are expressed in terms of the maximum observed signal, which was arbitrarily set at 100%. The improved protein microassay described in this paper was carried out as follows. BSP was added to citrate-HCl buffer (3) at a concentration of 60 &ml. Protein samples were prepared in 0.06% (v/v) Triton X-100 (Packard Instrument Co.) without prior treatment with NaOH, and 10 ~1 of each sample was added to duplicate microcentrifuge tubes containing 50 ~1 of the BSP reagent. Thus, the final concentration of Triton X-100 was 0.01% (v/v). All tubes were mixed, capped, and allowed to incubate overnight at room temperature. The BSP-protein aggregates were then precipitated by centrifugation, and the supernatant was carefully withdrawn and discarded. Several microliters of supernatant (-2 mm of fluid) was left in the bottom of each microcentrifuge tube in order not to disturb the BSP-protein precipitate. These pellets were washed twice with 250 ~1 of citrate buffer. A 75-~1 volume of 0.2 N NaOH was added with mixing to each pellet, and the amount of BSP which coprecipitated with the protein was quantified by determining the optical density at 580 nm. The optical density was measured by use of a Gilford Model 240 spectrophotom-

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eter (Oberlin, Ohio) with its standard pinhole aperture and a set of microcuvettes in an adjustable microcuvette carrier (Models 1012 and 1042A; Pyrocell Mfg. Co., Westwood, N. J.). All unknown protein solutions were assayed at two dilutions, for reasons given in the Results section. The polypropylene microcentrifuge tubes used in the new microassay were siliconized using a vaporizable silicone compound (Dri-Film SC-87; Pierce Chemicals, Rockford, Ill.). The tubes were placed in a vacuum chamber (Nalge, Rochester, N. Y.) with 0.5 ml of Dri-Film and subjected to a partial vacuum (-250 mm Hg) for 5 min. When atmospheric pressure was restored, the inrush of air carried the vaporized molecules of DriFilm into each tube, uniformly coating all surfaces. This cycle was repeated once, and the treated tubes were rinsed with water and allowed to dry. Bovine serum albumin (Fraction V, 96-99% pure) was purchased from Sigma Chemical Co., St. Louis, MO. The concentration of the standard albumin solution was calculated by use of the extinction coefficient of albumin at 280 nm (el $$ = 6.6; Ref. 10). Collagen was prepared from rat tails as previously described (5,6). The protein content of the collagen solution was determined by the biuret method (10). All measures of variability stated in this paper are standard errors of the mean. The number of samples (n) is given in parentheses. The Student’s t test was used to determine the significance of differences. A difference was considered statistically significant when p 5 0.02. RESULTS Evaluation

of the Nayyar

and Glick Protein Assay

Aliquots containing known amounts of bovine serum albumin were assayed using BSP as described by Bonting and Jones (8). This procedure measures the loss of BSP from the supernatant after precipitation of BSPprotein complexes. The results shown in Fig. 1 demonstrate that the optical density at 580 nm (OD5& of the supernatant decreased in direct proportion to added albumin in the 1- 10 pg range, as previously reported (8). However, the OD,,, of the supernatant was found to increase as the amount of protein was further increased in the 20- 100 pg range (Fig. 1). The resulting biphasic curve necessitated that unknown protein samples be assayed at two dilutions in order to determine whether the ascending or descending portion of the curve should be used to quantify the protein content. This biphasic curve might result from either decreased binding of BSP to protein or decreased aggregation of BSP-protein complexes at high protein concentrations. Equilibrium dialysis was used in order to determine the amount of BSP

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FIG. 1. Standard curve for assay of bovine serum albumin by the BSP dye-binding method developed by Nayyar and Glick (3). The method was as described by Bonting and Jones (8) except for minor modifications. Data between 0 and 20 pg of protein were analyzed by linear regression. The slope was -0.054 absorbance units/pg of protein, the intercept was 0.955 absorbance units, and the correlation coefficient was -0.99.

bound to protein at various protein concentrations. Dialysis curves corresponding to BSP solutions containing 0,20, and 100 Fg of albumin in the standard volume used by Bonting and Jones (70 ~1; Ref. 8) are shown in Fig. 2. These data demonstrate that more BSP is bound by 100 pg than by 20 pg of albumin (7.2 vs 6.4 pg). Thus, BSP still binds to protein at high protein concentrations even though the BSP dye-binding assay behaves as though very little protein were present. Light scattering was used to quantify the extent of aggregation of the BSP-protein complexes at different protein concentrations. The data obtained for the amount of light scattered form a biphasic curve when plotted against protein concentration (Fig. 3). This curve is inversely related to that shown in Fig. 1 for the BSP dye-binding assay. These data demonstrate that aggregation of BSP-protein complexes, as assessed by light scattering, initially increased with protein content, reached a peak at 25 pg, and then decreased by more than three orders of magnitude as the protein content increased from 25 to 100 pg. This decreased aggregation at high protein concentrations most likely explains the results obtained with the BSP dye-binding assay (Fig. l), since the BSP-protein complexes must precipitate in order to be separated from the unbound BSP. According to Oh et al. (4), collagen is not detected by the BSP dye-binding assay. Equilibrium dialysis was used in order to determine whether

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2. Equilibrium dialysis of BSP-protein mixtures. The optical density at 580 nm is a measure of the amount of unbound BSP released into the bathing solution after various periods of dialysis (see Methods). The curves are labeled according to the amount of protein initially present in 704 of the BSP-protein mixture (the same volume used in the BSP dyebinding assay shown in Fig. 1). Thus, the curves labeled 20 and 100 wg of albumin correspond directly to the same values shown in Fig. 1. FIG.

FIG. 3. Light scattering by BSP-protein mixtures. Measurements were made as described in Methods. Protein values are given in micrograms per 70 ~1 of solution and are therefore directly comparable to those shown in Fig. 1 and 2.

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BSP binds to collagen. The results shown in Fig. 2 demonstrate that collagen binds only 9% as much BSP as an equivalent amount of albumin, which almost certainly explains why it is not detected by BSP dye-binding assays. Evaluation of a Micromethod for Protein Analysis Using BSP

The BSP protein assay proved to be unsatisfactory in the O-2 pg range because the signal-to-noise ratio was too small. For example, the range of optical density of the blanks shown in Fig. 1 was 0.106, while the change in optical density for 1 pg of protein was calculated to be 0.054. With such a small signal-to-noise ratio, an accurate protein measurement can not be made unless large numbers of replicate samples are assayed. A sensitive new microassay for protein has been developed that differs from the method described by either Nayyar and Glick (3) or Bonting and Jones (8) in three important respects. First, the new assay is similar to that of Greif (1 l), in that it quantifies the amount of BSP coprecipitated with the protein rather than the amount left in solution. This approach yields a very low, consistent blank (0.006 + 0.001, n = 8) and thus partially overcomes the source of error discussed above. Second, the entire amount of precipitated BSP is resuspended in the minimum volume that can easily be examined in a normal spectrophotometer (-75 ~1). Thus, the change in

FIG. 4. Effect of Triton X-100 on the amount of BSP precipitated per microgram of bovine serum albumin. The microassay was carried out as described in Methods. Each sample contained 1 pg of protein, and the final Triton X-100 concentration in the reaction mixture was varied as indicated on the abscissa. All plotted points represent means 2 SEM (n = 4).

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FIG. 5. Standard curves for bovine serum albumin and collagen as determined by the BSP microassay. Details of the procedure are given in Methods. Data between 0 and 5 pg were analyzed by linear regression. The slope was 0.449 absorbance unitsipg of protein, the intercept was 0.028 absorbance units, and the correlation coefficient was 0.99.

OD,,, per microgram of protein is increased. Third, the assay is carried out in the presence of 0.01% Triton X-100, which was serendipitously found to enhance the amount of BSP precipitated per microgram of protein (Fig. 4). Other technical details are given in Methods. Data obtained using the new microassay demonstrate that this procedure is linear from 0.1 to 5 pg of albumin (Fig. 5). In addition, it is almost an order of magnitude more sensitive than the Nayyar and Glick method as described by Bonting and Jones (+0.449 vs -0.054 absorbance units/pg of protein). On the other hand, this microassay is identical to that of Nayyar and Glick (3) in two important aspects. First, it does not detect collagen (4). According to the data shown in Fig. 5, the microassay is 90-fold more sensitive for albumin than for collagen. Second, the curve relating color yield to protein concentration is biphasic (cf. Figs. 1 and 5). Thus, protein samples must still be assayed at two dilutions. The BSP microassay can be employed to quantify protein in dissociated cell cultures grown on a collagen substrate (12). Data obtained using sympathetic ganglion cultures grown on a layer of collagen are shown in Fig. 6. A blank value of 0.7 t 0.3 (n = 6) pg of protein (9 pg/well) was obtained in the presence of collagen alone.’ Addition of sympathetic ganglion ’ On the basis of the results presented in Fig. 5, a blank value of approximately 0.1 pg of protein might be expected. The extra protein detected in the blanks is probably due to attachment of proteins in the culture medium to the collagen surface. For example, up to 2 pg of protein can adhere to a polystyrene surface of equal size after exposure to culture medium containing 10% fetal calf serum.

WALLACE

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FIG. 6. Determination of protein in dissociated sympathetic ganglion cell cultures by the BSP microassay procedure. Various numbers of dissociated cells from sympathetic ganglia excised from 1Zday chick embryos were plated on collagen and grown in vitro for 96 hr (12). The cultures were rinsed three times, harvested by sonication in 0.06% Triton X-100, and assayed as described in Methods. Each culture well contained 9 pg of collagen, which had been precipitated to form a substrate for cell adhesion and growth (12). All plotted points represent means 2 SEM (n = 6).

cells resulted in a linear increase in the total amount of protein detected (Fig. 6). Analysis of these data by linear regression using bovine albumin as the reference standard yields a slope of 77 + 10 pg of BSP-reactive protein per ganglion cell. However, the proteins in the ganglion cells almost certainly are not equivalent to bovine albumin in their relative content of BSP-binding cationic groups. Data presented by Nayyar and Glick (3) demonstrate that bovine albumin is approximately 23% more reactive on the basis of protein nitrogen than the protein found in a number of other tissues. Thus, the corrected protein value should be approximately 95 pg of protein per ganglion cell. Similarly, ganglion cells were found to contain 97 & 3 (n = 5) pg of protein when identical cultures were plated on a polystyrene surface and assayed by the Lowry method (10). Thus, the values obtained by these two protein assays are not significantly different. DISCUSSION

A BSP dye-binding microassay for protein, which should be especially useful to those interested in quantifying cellular protein in cultures grown on collagen, has been developed. This assay is about eight times as sensitive as most other BSP dye-binding procedures (3,8,9). The useful range of our assay is from 0.1 to 5.0 pg of protein, while other workers have reported linearity in the range of l-10 (8), 1.4-4.9 (12), and 0.5-2.25 pg of protein nitrogen (approximately 3-14 pg of protein; Ref. 3). Our greater

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sensitivity is largely due to the use of Triton X-100 to enhance dye-binding by protein (Fig. 4). In contrast, Nayyar and Glick (3) achieved a similar degree of sensitivity (22-130 ng of protein nitrogen, or approximately 0.14-0.8 pg of protein) by significantly reducing the volumes at each step and determining optical density by use of a microscope photometer and capillary cuvettes. Data presented in this paper demonstrate that high concentrations of protein can reduce the efficiency of aggregation of BSP-protein complexes (Fig. 3). The resulting biphasic precipitation curves are strikingly similar to immunoprecipitin curves obtained for antigen-antibody interactions (13). Equilibrium dialysis shows that protein continues to bind BSP at high protein concentrations (Fig. 2) in spite of the decreased efficiency of aggregation. Maximal precipitation occured at BSP/protein molar ratios of 34 and 27 for the BSP dye-binding technique and the new method, respectively. This suggests that the efficiency of coprecipitation of the BSP-protein complexes decreases whenever less than -30 BSP molecules are bound per albumin molecule. To our knowledge, this is the first report of such anomalous behavior in a dye-binding assay at high protein concentrations. The binding of 30 mol of BSP/mol of albumin observed in the present study is somewhat greater than the value of 10 mol/mol of protein determined by Piller (14). Differences in the conditions under which binding was measured (i.e., bovine albumin at pH 2 vs human albumin at pH 7) probably account for the observed difference in the level of saturation of BSP binding to albumin. Collagen was almost undetectable using the new micromethod (Fig. 5); this confirms an earlier report by Oh ef al. (4) using the method of Nayyar and Glick (3). Equilibrium dialysis demonstrated that collagen bound only 9% as much BSP as an equal weight of albumin (Fig. 2). The greatly reduced BSP binding probably explains why collagen cannot be quantified by this procedure. ACKNOWLEDGMENTS We would like to thank Dr. Dixon M. Woodbury for careful reading of this manuscript. This research was funded by U. S. Public Health Service Grants NS-12812 and GM-00153 and by a grant from the Epilepsy Foundation of America.

REFERENCES 1. Glick, D. (1963) Quantitative Chemical Techniques of Histo- and Cytochemistry, Vol. 2, pp. 148-150, Wiley-Interscience, New York. 2. Glick, D., Good, R. A., Greenberg, L. J., Eddy, J. J., and Day, N. K. (1958) Science 128, 1625-1626. 3. Nayyar, S. N., and Glick, D. (1954) J. Histochem. Cytochem. 2, 282-290. 4. Oh, T. H., Kim. S. U., and Johnson, D. D. (1975) Neurobiol. 5, 188-191.

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5. Bomstein, M. B. (1958) Lab. Invest. 7, 134-137. 6. Ehrmann, R. L., and Gey, G. 0. (1956) J. Nat. Can. Inst. 16, 1375-1403. 7. Hauschka, S. D., and Konigsberg, I. R. (1966) Proc. Nat. Acad. Sci. USA 55, 119-126. 8. Bonting, S. L., and Jones, M. (1957) Arch. Biochem. Biophys. 66, 340-353. 9. Diamant, B. Redlich, D., and Glick, D. (1967) Anal. Biochem. 21, 135-146. 10. Rutter, W. J. (1967) in Methods in Developmental Biology (Wilt, F. H., and Wessells, N. K., eds), pp 675-677, Thomas Y. Crowell, New York. 11. Greif, R. L. (1950) Proc. Sot. Exp. Rio!. Med. 75, 813-815. 12. McCarthy, K. D., and Partlow, L. M. (1976) Brain Res. 14, 391-414. 13. Eisen, H. N. (1974) Immunology, pp. 370-376, Harper and Row, New York. 14. Piller, M. (1963) Schweiz. Med. Wochschr. 93, 1034- 1038.

A sensitive microassay for protein in cells cultured on collagen.

ANALYTICAL BIOCHEMISTRY 87, l- 10 (1978) A Sensitive Microassay for Protein Cells Cultured on Collagen in L. J. WALLACE AND L. M. PARTLOW Departm...
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