Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye

AS.ALTTICAL

BIOCHEMISTRY

Differential

56, 43-51

Staining Gels

with

M. R. GREEN,

of Phosphoproteins a Cationic

on Polyacrylamide

Carbocyanine

,J. V. PASTEWKA,

Chemistry National I&it&s Received

(1973)

Dye

A. C. PEACOCK

AND

Branch, National Cancer Institute, of Health, Bethesda, Marvlaud March

8, 1973;

accepted

May

20014

4, 1973

INTRODUCTION

The absorption spectrum of the cationic carbocyanine dye 1-ethyl-2] 3- (1-ethylnaphtho [ 1,2d] thiazolin-2-ylidene) -2-methyl-propenyl] -naphtho[ 1,2d] thiazolium bromide in solution as a function of solvent, temperature, pH, dye concentration and inorganic ion concentration was studied in detail by Kay et al. (1). Interaction of the dye with trace amounts of proteins, nucleic acids and substituted polysaccharides caused changes in the absorption spectrum of the dye. At least five different complex states of polymer-dye interaction were found (2). The complex states and corresponding spectral band maxima were: a state (570 nm), /3 state (535 nm), y state (50&510 nm), J state (620-650 nm), ,&x state (550 nm, a hybrid or mixture) and the S state (470 nm). The states depended on the adsorption of the dye by the macromolecule and on the nature and conformation of the macromolecule. Dahlberg et al. (3) used this dye as a sensitive stain for nucleic acids electrophoresed on polyacrylamide gels. They called it “Stains-all” because it stained RNA (bluish purple), DNA (blue), protein (red) and acid mucopolysaccharides (various hues). Bader et al. (4) explored further the use of this stain for quantitation of hyaluronic acid and noted differences in staining properties between this substance and the chondroitin sulfates and heparin following polyacrylamide gel eleetrophoresis. We now report that an additional class of compounds, the phosphorylated proteins such as milk casein, can be detected by use of this dye. Such proteins stain blue, in contrast to nonphosphorylated proteins which stain either red or are stained not at all. MATERIALS

AND

METHODS

The I-ethyl-2- [3- (I-ethylnaphtho ]1,2d] thiazolin-2-ylidene) -2-methylpropenyl] -naphtha [ 1,2d] thiazolium bromide was obtained from Eastman Copyright All rights

43 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.

44

GREEN,

PASTEWKA,

AND

PEACOCK

Organic Chemicals and prepared as a 0.1% stock solution in formamide according to the procedure of Dahlberg et al. (3). The working stain was prepared just before use by combining 10 ml of the stock stain, 10 ml formamide, 50 ml isopropanol, 1 ml 3.0 M Tris-HCl pH 8.8 and deionized water to a volume of 200 ml. The bovine aSI-casein, /?-casein AZ, K-casein and a-lactalbumin were gifts from the Department of Agriculture, Eastern Utilization Research and Development Division. Pepsin, ovalbumin and bovine albumin were obtained from Sigma, Hammersten casein from Mann Research Laboratories and phosvitin from Schwara/Mann. Milk and mammary explants were from Balb/C mice. Electrophoresis and staining. The purified proteins, mouse milk and homogenates of mouse mammary explants were electrophoresed at pH 4.0 on 10% polyacrylamide cylindrical gels containing 8 M urea, 2.5 mA per gel (5,6) ; or at pH 7.2 on 7% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), 5 mA per gel (5). SDS interfered with the staining. It was removed from the gels by agitation in 25% isopropanol (7), 30 ml per gel, at 50°C for 15 min. The acid-urea gels did not require this pre-treatment. The gels were rinsed and placed in the staining solution in the dark overnight. They were rinsed with several changes of deionized water and destained in deionized water in the dark or shielded from direct light until a good contrast between the bands and background was observed. When exposed to direct light the gel background became a muddy yellow. Scanning was performed on a Gilford spectrophotometer, Model 240, equipped with a linear transport and recorder. The stained proteins were scanned at 20-nm intervals from 480 to 700 nm; readings were made at 5-nm intervals in the regions of maximum absorptions. The absorbance at each wavelength was plotted to obtain the spectral absorption curves of the proteins studied. The aSI-casein, scanned on 3 consecutive days, showed no significant change in absorbance or shift in optimal wavelength. Gels were photographed using a Polaroid MP-3 Land Camera wit.h a red filter. The color photography using Ektachrome was done by the NIH Photography Section of Medical Arts and Photography Branch. Phosphorus analysis. Samples for phosphorus determination were digested (8), and phosphorus determined following Allen’s (9) procedure with the modification that 5 N H,SO, was substituted for 60% per&lo& acid. Enzymatic dephosphorylation. Alkaline phosphatase (highly purified Type VII) from calf intestinal mucosa (380 units/mg protein) was obt,ained from Sigma. Incubation conditions were similar to those of

STAINIKG

OF

PHOSPHOPROTEINS

45

Kalan and Telka (10). The cYS1-casein was incubated with the alkaline phosphatase (1O:l) (wt/wt) for 2 and 20 hr at 37°C in 0.005 M Na acetate buffer, pH 5.5 containing 0.001 M MgCI,. The solution was adjusted to a final pH of 6.2 with NaOH. Dephosphorylation was nearly complete at 2 hr. RESULTS

We wished to determine how the phosphorus content of a protein influenced the absorption maxima of the dye-protein complex. Protein samples wit,h phosphorus content (verified by direct analysis) ranging from 0 to 10% were used. They were electrophoresed in the acid-urea gels and spectral absorptions of the resulting patterns were taken at 20-nm intervals from 480 to 700 nm. The nonphosphorus (bovine albumin) and lo,w-phosphorus (pepsin) containing proteins had absorption maxima between 500-520 nm and stained red. The K-casein, 0.2% P, and proteins with a greater phosphorus content had absorption maxima in the 640-660 nm region and stained blue (Figs. 1 and 2). The absorption maximum of phosvitin was 640 nm; the absorption maximum of &,-casein and the major band of the p casein was at, 660 nm. By eye, we were able to identify Hammersten casein (Fig. 3, bottom) aSI-casein and /3-casein A? as phosphoproteins at concentrations as low as l-2 pg protein. We could not identify K-casein unless the concentration was about 5 pg. Under our acid-urea conditions, Hammersten and p caseins were electrophoretically heterogeneous. Hammersten casein produced two major and several minor bands (Fig. 2). The latter could not be seen with the Amidoschwarz stain. We determined the effect, of varying protein concentrations of caseins at 660 run. Hammersten, & and p-caseins in concentrations ranging from 2 to 10 pg were applied to gels for electrophoresis. Absorbance at 660 nm of the major peaks on each casein was plotted as a function of the concentra.tion of the protein applied. Figure 4 shows the linear relationship between absorbance and concentration. The range over which this relationship was linear depended on t,he phosphorus content of the protein. Dephosphorylation of as,-casein with alkaline phosphatase, caused a diminution of the major peak and the emergence of proteins having both slower and faster mobilities than the original peak (Fig. 5, left panel, Amidoschwarz stain). A similar result was found with a duplicate gel st,ained wit’11 “Stains-all” but scanned at 520 nm (red component,). A different result was found when the “Stains-all” gel was scanned at 660 nm (blue component) (Fig. 5, right panel). The absorbance of &I,-

46

GREEN,

PASTEWKA,

AND

PEACOCK

14-

IZ-

_------. -I-------

Phawfm ] 0 SI -Carein, Bowne B- Cosein AZ, Bowne K-Cosem, Ewne Pepsin Albumir, Bovme

21/2/4

1.0 -

460

520

560

600

WAVELENGTH

640

680

I 720

(pm)

FIG. 1. Spectral absorbance curves of “Stains-all” stained proteins electrophoresed at acid pH with 8 M urea were taken at 20-nm intervals from 480 to 700 nm. The phosphorus content of the proteins was checked by analysis and per cent values given in the literature were confirmed: &-casein, bovine 1.0; p-casein A*, bovine 0.6; K-casein, bovine 0.2 (11); pepsin 0.09; phosvitin 8-10; (12); albumin, bovine
FIG. 2. (Top to bottom) : pepsin (10 pg) red, phosvitin, (5 w) blue, electrophoresed at acid pH, S-M urea.

,8-casein A* and c&L-casein

STAINING

FIG.

albumin mer&en

OF

PHOSPHOPROTEINS

3. Proteins electrophorcsed at acid pH, 8 M urea. Top two (6 gg) : a, “Stains-all” (red) ; b, Amidoschwarz. Two bottom casein (8 pg) : c, “Stains-all” (blue) ; d, Amidoschxarz.

1.6

gels-bovine gels-Ham-

r

1.2 ,&Casem

A2 Peak- I

P Hommersten

0

2

4

6

Peak - I

,AHammerslen

Peak -2

0 @Case,n

AZ Peak - 2

I

a

,~g PROTEIN

FIG. 4. Absorbance at 660 nm as a function of concentration of caseins. Heights of major peaks in Hammersten casein and p-casein were plotted at the various concentrations of total protein applied to the acid gel. &XL-Casein had only one ma,ior peak at 660 nm.

GREEN,

PASTEWKA,

AND

PEACOCK

0.6n

05-

0.4

-

0.3

-

: 5 9 :: 2

02-

FIG. 5. Electrophoresis at, pH 4.0 on a 10% polyacrylamide gel containing 8 M urea. Scans of a& casein, 0.9% P (-) and dephosphorylated cu&-casein, 0.08% P (- - - - -). Panel A : Amidoschwarz scanned at, 603 nm. Panel R : “Stains-all” scanned at 660 nm.

in the main peak was three times greater than with Amidoschwarz and was virtually lost upon dephosphorylation. The relative migrations (Rm) of t,he peaks were determined with respect to a tracking dye. In order to test the applicability of this stain to distinguish phosphorylated proteins from other proteins, we used mouse milk. This presented a natural source of an unknown solution. Mouse milk was electrophoresed at pH 7.2 on an SDS gel and stained with Amidoschwarz (Fig. 6a) or “Stains-all” (Fig. 6b). The blue staining bands in Fig. 6b correspond to the caseins. Caseins precipitate following acidification of milk at pH 4.6 or following treatment with Ca2+ and rennin at pH 6.7. All of the blue staining bands present in the milk were found in the precipitated caseins. The number of casein bands which could be seen depended on the electrophoretic conditions. In the acid-urea system (Fig. 7) there were fewer bands than in the pH 7.2-SDS system. One of the whey proteins in the 13,600 g supernatant, stained red, the fastest migrating protein did not stain (compare A-3,4 with B-3,4). Dephosphorylation of the milk with alkaline phosphatase resulted in a loss of the blue color in

casein

STAIKISG

OF

49

PHOSPHOPHOTEIKS

FIG. 6. Mouse milk electropboresed at pH 7.2 in a 0.1% SDS-7% acrylamide gel. a, Amidoschwarz: b. ‘Stains-all (blue). The band at 60-62 mn in (a) ia an insulin marker added to the milk solution. This marker as well as some other milk proteins did not stain blue x-it11 “Stains-all” (h). A filter was used to eliminate the red bands for photography.

the casein region A-4. The level of alkaline phosphatase used was such that there was no shifting of position nor loss in the non-phosphorus containing proteins (Fig. 7B, 3 and 4). A mixture of proteins from a supra-mitochondrial supernatant of mid-

(:I::::

1

FIG. 7. Electrophoresis of mouse milk at acid pH. 8 M urea, “Stains-all”; B. Amidoschwarz; 1, whey proteins; 2. casein-:: milk dephosphorylated with alkaline phosphatase.

1

6 cm

10% ao,~lnmide 3, mouse milk;

gel. X. and 4,

50

GREEN,

PASTEWKA,

AND

PEACOCK

pregnant mouse mammary gland homogenate was electrophoresed in the acid-urea system (5). In this case also, the caseins were identifiable as blue staining bands with “Stains-all.” DISCUSSION

Use of the carbocyanine dye “Stains-all” enables one to distinguish certain phosphoproteins from non-phosphorylated proteins. The former stain blue; the latter stain red or remain unstained. While other macromolecules such as nucleic acids or mucopolysaccharides have also been shown to stain shades of blue or purple, these may be distinguished from the blue staining phosphoproteins by prior chemical fractionation, differential centrifugations, specific enzymatic digestions or by exclusion from low porosity gels. Phosphoproteins electrophoresed at acid pH did not stain with the original formulation used for staining nucleic acids on gels. Several changes were made. Tris buffer at pH 8.8 was incorporated in the solution, isopropanol was included as a fixative and the concentration of formamide was decreased. Absorption at 640-660 is dependent on the interaction of phosphorus with the dye. Kay (13) noted that the effect of pH on complexing reactions with macromolecules other than proteins was small. No polymer containing only phosphate anionic sites was found specifically to form either the J complex or the S complex. Polyphosphate complexes were relatively independent of pH (2). The influence of pH on the dye-protein complexes is attributed to changes in the net charge of the protein. At pH values above the iso-electric point, of a particular protein, the net charge is negative and more of the cationic dye interacts with the protein. The reaction of t.he dye with phosphoproteins may not solely depend on the phosphorus content but also on the spacing of the phosphorus molecules. Peptide analysis of partial hydrolysates of the caseins, phosvitin, and protamine show that phosphoseryl residues occur in blocks of up to four residues in a row (1618). Based on studies of the dye-macromolecule interaction in solution, Bean et al. (2) concluded that the J state (absorption at 620-660 nm) is due to the reaction of individual dye molecules at isolated sites. These authors stated that., “In addition, it appears desirable to assume that the spectra of some of the other complex states (at other wavelengths) may be caused by specific interaction of the dye with the polymer rather than by dye-dye interactions between adjacent dye molecules on the polymer.” They found that, anionic sites in a polymeric matrix were more effective than the corresponding simple anions in creating specific

STAINIKCi

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51

PHOSPHOPROTEINS

complexes with the stain. The effect of spacing of anions by methylation of anionic groups in pectic acid and pectins was investigated. As the average spacing ,between anionic groups increased, t.here was a shift from the (Y band (560-572 nm) to the J band. Further studies, concerning the spacing of phosphorus in phosphoproteins and the interaction of these proteins with the dye seem warranted. ADDENDUM Prior to stainin@~, gels electrophoresed at alkaline pH were pre-treated 25% isopropanol in the same manner as the SDS containing gels.

with

ACKXOWLEDGMENT We thank Dr. A. T. Ness for helpful

discussions and suggestions.

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13.

10, 2606. P. B., OSER, B. L., AND SUMMERSON, N. H. (1947) in Practical Physiological Chemistry, 12th Ed., The Blakiston Co. ALLEN, R. J. L. (1940) Biochem. J. 34, 858. KALAN, E. B., AND TELKA, M. (1959) Arch. Biochem. 85, 273. THOMPSON, M. P., JARASUK. N. P., JENNERS, R. LILLEVIK, H. A., ASHWORTII, U. S., AND ROSE, D. (1965) J. Dairy Sci. 48, 159. PERLMAN, G. E. (1955) Adv. Prot. Chem. 10, 1. KAY, R. E., WALWICK, E. R., AND GIFRORD, C. K. (196413) J. Phys. C&m.

14. 15. 16. 17. 18.

68, 1907. MANSON, W., AND ANNAN, W. D, (1971) Arch. Biochem. Biophys. 145, SANDERS, M. M., AND DIXON, G. H. (1972) J. Bid. Chem. 247, 851. MANO, Y., AND LIPMANN, F. (1966) J. Rio/. Chem. 241, 3822. WILLIAMS, J., AND SANGER, F. (1959) Biochim. Biophys. Acta 33, 294. CLARK. R. C. (1972) Comp. Biochem. Physiol. 41B, 891.

8. HAWK, 9.

10. 11. 12.

16.