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Purification and some properties of the phosphoprotein from rat incisors
Biochimica et Biophysica Acta, 427 (1976) 262-276
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37274 P U R I F I C A T I O N A N D SOME P R O P E R T I E S OF T H E P H O S P H O P R O T E I N FROM RAT INCISORS
WILLIAM T. BUTLER, WILLIAM T. HALL and WILLIAM S. RICHARDSON Institute of Dental Research, University of Alabama in Birmingham, University Station, Birmingham, Ala. 35294 (U.S.A.)
(Received August 13th, 1975)
SUMMARY The phosphoprotein of rat incisors has been purified by successive gel and ion-exchange chromatography. The product gave a single band on polyacrylamide gel electrophoresis and contained approximately 3 4 ~ phosphoserine and 3 2 ~ aspartic acid. Alkaline elimination experiments showed all the phosphate to be present as phosphoserine. Ultraviolet spectra in the presence or absence of ATP showed that the phosphoprotein did not contain a nucleotide moiety as suggested by Veis, A., Spector, A. R. and Zamoscianyk, H. ((1972) Biochim. Biophys. Acta 257, 404-413) for bovine dentin phosphoprotein.
INTRODUCTION One of the organic constituents of dentin is a polyanionic protein containing high levels of phosphate, serine and aspartate. In the unerupted bovine molar this phosphoprotein is only partially soluble and is believed to occur in two forms: insoluble, covalently bound to collagen [1] and soluble, conjugated to a nucleotide like component [2, 3]. In contrast, the rat phosphoprotein is almost completely soluble [4]. This report summarizes a procedure for purifying the rat phosphoprotein and presents evidence that the phosphoprotein from this species does not occur conjugated to a nucleotide. MATERIALS AND METHODS Extraction and purification
The phosphoprotein was extracted from rat incisors essentially as described [4]. The extract was desalted on a column of Bio-Gel P-2 (Bio-Rad, 100-200 mesh) and eluted with 0.2 M acetic acid and lyophilyzed. A 20 mg sample was dissolved in 5 ml of 0.01 M Tris-HC1 buffer (pH 7.4) and placed on a 2.5 x 60 cm column of Sephadex G-150 (Pharmacia, medium) and eluted with the dissolving buffer (Fig. l). The effluent was monitored at 230 nm with a Beckman DB-G spectrophotometer equipped with a flow cell. Fractions of 5 ml were collected and aliquots were assayed for phosphate liberated after hydrolysis
263 with 0.2 M N a O H at 37 °C for 24 h. Phosphate analyses were performed with a Technicon Auto-Analyzer [5]. Phosphate-containing fractions from the Sephadex G-150 column were pooled and directly applied to a 2.0 × 20 cm column of DEAE-cellulose (Whatman DE-32, microgranular) previously equilibrated with 0.01 M Tris-HC1 buffer (pH 7.4). It was eluted with a linear gradient from 0 to 1.0 M NaC1 over total volume of 1 liter (Fig. 2). The effluent was monitored as described in the preceding paragraph. Phosphoprotein tractions were desalted as described above and lyophilyzed.
Analysis Disc gel electrophoresis was by the method of Davis [6] with 7 ~ polyacrylamide gels. Samples of 0.2 to 0.3 mg were applied and, after runs of 45 min at 7 mA per tube, the gels were stained with Coomassie Blue [7] and destained by diffusion in 20 ~ acetic acid. For amino acid and phosphate analysis of the purified phosphoprotein, approximately 2-rag samples, previously dried in vacuo over Drierite, were hydrolyzed in constant boiling HC1 at 108 °C for 24 h. Hydrolysis was under reduced pressure in a vacuum desicator which was saturated with 6 M HC1 [8]. Both analyses were performed on aliquots of the same hydrolyzates. Amino acid analysis was with a Beckman 120C analyzer modified for single-column analysis [9] (Table I). Analyses foi phosphate were as outlined above; results were compared to standards containing 0.05 to 0.80 #mol per ml NaH2PO4" H20. The observed amount of serine measured on the amino acid analyzer arises from seryl and phosphoseryl residues which are destroyed by acid hydrolysis at different rates. Since the phosphoserine value was known from the measured phosphate and, assuming that 10 ~ of the serine [10] and 25 ~o of the phosphoserine [11] were destroyed, the amount of serine could then be calculated from the algebraic expression : Serob~ = 0.90 Ser + 0.75 Psr where Serobs represents the amount of serine measured with the amino acid analyzer. To determine if all the phosphate of the phosphoprotein were present as phosphoserine, the protein was subjected to an alkaline elimination reaction. The amount of serine lost was compared to the amount of inorganic phosphate liberated. Duplicate samples were incubated at 37 °C in 0.2 M N a O H alongside controls incubated in water. Aliquots were then taken for amino acid and phosphate analyses. Analysis for calcium was performed by Galbraith Laboratories, Inc., Knoxville, Tennessee.
Ultraviolet spectral analysis The spectra of solutions of the purified phosphoprotein and of the phosphoprotein containing ATP were recorded with a Beckman Acta III recording spectrophotometer. Solutions containing 1 mg/ml and 0.125 mg/ml of phosphoprotein in 0.05 M sodium phosphate buffer (pH 7.0) in 1 cm cuvettes were initially used. Appropriate aliquots of a 1 mg/ml solution of ATP in the same buffer were added and the spectra recorded again.
264 RESULTS
Gel chromatography of the crude phosphoprotein resulted in a single major peak eluting in the void volume of the column (Fig. 1). The phosphate closely followed this excluded peak. Subsequent chromatography of the phosphoprotein on DEAEcellulose (Fig. 2) resulted in a single major peak eluting at approximately 0.4 M NaC1. Again, the phosphate corresponded closely to the ultraviolet absorption for the major peak. The resultant phosphoprotein was judged to be pure, since a single protein band was observed when the material was subjected to polyacrylamide gel electrophoresis.
14-
o
12 ~
I~l I003
&
O.2-, 50
O0
2OO
150
250
300
EFFLUENT VOLUME, ml
Fig. 1. Gel filtration o f crude rat incisor phosphoprotein. A 20 m g sample was applied to a 2.5 × 60 c m c o l u m n o f Sephadex G-150 (medium) e m p l o y i n g 0.01 M Tris-HC1 (pH 7.4) as elution buffer. The effluent was monitored for protein at 230 n m and for phosphate after treating aliquots o f the 5-ml fractions with 0.2 M N a O H for 24 h at 37 °C. Phosphate analyses were performed with a Technicon Auto-Analyzer [5]. T 0.6
E 0 o,4
0.2
o,O~ 0.0.
O0
ioo
200
300
400
500
EFFLUENT VOLUME, ml
Fig. 2. Ion-exchange chromatography of phosphoprotein. Appropriate fractions from Sephadex G 150 (Fig. 1) were pooled and directly applied to a 2.0 × 2 0 c m c o l u m n of DEAE-cellulose ( W h a t m a n DE-32, microgranular) previously equilibrated with 0.01 M Tris-HCI (pH 7.4). Elution was with a linear gradient f r o m 0 to 1.0 M NaC1 over a total volume o f I liter. The effluent was monitored as described in the legend o f Fig. 1. Phosphoprotein fractions were desalted on a column of Bio-Gel P-2 and lyophilized.
The composition of the purified phosphoprotein (Table I) was similar to that reported for other dentinal phosphoproteins [2, 12]. Phosphoserine and aspartic acid accounted for 65 % of the residues of the protein. About 80 ~ of the weight of protein hydrolyzed in these experiments could be recovered as amino acids. Phosphoseryl residues were assumed to be in the monosodium form for these calculations. The calcium content was only 0.32 ~ 0.0l %.
265 TABLE I AMINO ACID COMPOSITION OF RAT INCISOR PHOSPHOPROTEIN Amino a c i d
Residues/thousand
Phosphoserine Aspartic acid Serine Glutamic acid Glycine Threonine Alanine Lysine Proline Histidine Arginine Half cystine Leucine Valine Isoleucine Tyrosine Phenylalanine
339.0 315.0 170.0 50.1 32.4 17.9 15.4 11.4 8.1 7.0 7.0 6.9 5.8 5.2 3.8 3.1 1.6
The amount of serine destroyed by treatment of the phosphoprotein with mild alkali corresponded closely to the amount of inorganic phosphate liberated by this procedure. For example, in one experiment the loss of serine was 2.61, and the level o f nascent phosphate was 2.67/zmol per mg of sample. Threonine levels remained constant during the treatment. In view of the conclusion that the soluble phosphoprotein from bovine molars is conjugated to a nucleotide [2, 3], we decided to examine the effect o f various amounts of ATP upon the ultraviolet spectral properties of the rat incisor phosphoprotein. The spectrum of solutions containing 1 mg/ml or 0.125 mg/ml (Fig. 3) of the phosphoprotein showed no discrete absorption peaks in the 230 to 300 nm range, but a large absorbance peak at approximately 205 nm, the n -~ zt* transition of the t0 o9
C\ F
O.8 w07 0 Z 06
~04 m '~ 0.3 02 0.1 200
220
240
260
280
300
WAVELENGTH, nm
Fig. 3. Ultraviolet spectrum of rat incisor phosphoprotein ( - - - - - - , 0.125 mg/ml) and rat incisor phosphoprotein with 5 ~ ATP ( - - - - ,0.120 mg/ml protein and 0.006 mg/ml ATP).
266 peptide groups [13]. Upon addition of ATP equal to 5 ~ of the weight of the phosphoprotein a distinct absorbance peak at 260 nm was observed (Fig. 3). A slight increase in the 205 nm peak was also observed under these conditions. When the solution contained equal weights of ATP and phosphoprotein (0.060 mg/ml), both the 260 and 205 nm peaks were quite large. Control experiments and calculations utilizing the extinction coefficient of ATP showed that the presence of the phosphoprotein did not alter the level of the 260 nm absorbance of ATP. DISCUSSION Veis et al. [2] have reported that the phosphoprotein from bovine dentin is conjugated to a nonprotein moiety believed to be a "purine, an adenine derivative" [3]. The conclusion that a nonprotein substituent accounts for about 5 0 ~ of the weight of the phosphoprotein was based on the inability to account for the weight of the preparation as amino acids and the fact that the 260 nm absorbance of the protein was greater than that at 280 nm [2]. In addition, the total phosphorus found by elemental analysis was greater than could be accounted for as phosphoserine with the methods employed. In contrast, about 80 ~ of the weight of the phosphoprotein from rat incisors could be accounted for as amino acids. While the remainder of the weight was neither carbohydrate [12] nor calcium, it could have been tightly bound water or unknown metals. The alkaline elimination experiments clearly demonstrated that all the phosphate could be accounted for as phosphoserine. The ultraviolet spectral studies (Fig. 3) showed that the addition of A T P gave a discrete 260 nm absorbance peak not seen in the solutions containing only the phosphoprotein. Thus there is no evidence that the rat phosphoprotein contained a nucleotide, though the spectrum of the phosphoprotein would have ieadily revealed the presence of less than 5 ~ by weight of nucleotide (Fig. 3). If 50 ~o of the weight of the protein did indeed consist of a nucleotide, a spectrum with a strong 260 nm absorbance should have been obtained. Veis et al. [2] and Spector [14] have reported spectral data for the bovine phosphoprotein. The ultraviolet spectrum [14] displayed only slight absorbances at 260 nm and 280 nm and a strong absorbance below 230 nm. We evaluated this spectrum for nucleotide content, using the extinction coefficient of ATP at 260 nm, and for protein content reflected by the 230 nm absorbance (compared to that of a known amount of rat incisor phosphoprotein with a remarkably similaI amino acid content). We conclude that the bovine phosphoprotein could contain no more than 2 ~ by weight of nucleotide. In addition, the reported 260 nm to 280 nm ratio of the bovine phosphoprotein was approximately 2.0 [2, 14], indicating the possibility of high nucleotide content. However, Layne [15] reviewed the use of relative absorbances at 260 nm and 280 nm for the determination of pl otein in the presence of nucleotides. Only proteins with distinct 280 nm absorbances were candidates for the technique and, even then, the determinations had the potential for considerable error. We, thelefore, strongly suggest that, sir, ce the level of aromatic amino acids in the bovine phosphoprotein [2, 14] is extremely low and the 280 nm absorbance [14] is correspordingly weak, that a ratio of 260 nm to 280 nm is not a reliable indicator for the presence of a nucleotide.
267 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Veis, A. and Perry, A. (1967) Biochemistry 6, 2409-2416 Veis, A., Spector, A. R. and Zamoscianyk, H. (1972) Biochim. Biophys. Acta 257, 404-413 Sharkey, M. A. and Veis, A. (1974) J. Dental Res. 53 (Special Issue), 57-63 Butler, W. T., Finch, J. E. and DeSteno, C. V. (1972) Biochim. Biophys. Acta 257, 167-171 Technicon Auto-Analyzer Methodology (1968) Technicon Corp., Tarrytown, N.Y. Davis, B. T. (1964) Ann. N.Y. Acad. Sci. 121, 404-427 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 Dreyer, W. J. and Bynum, E. (1967) in Methods of Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. 11, pp. 32-39, Academic Press, New York Miller, E. J. and Piez, K. A. (1966) Anal. Biochem. 16, 320-326 Downs, F. and Pigman, W. (1970) Int. J. Protein Res. 2, 27-36 Allerton, S. E. and Perlman, G. E. (1965) J. Biol. Chem. 240, 3892-3898 Butler, W. T. (1972) in The Comparative Molecular Biology of Extracellular Matrices (Slavkin, H. C., ed.), pp. 255-259, Academic Press, New York Haschemeyer, R. H. and Haschemeyer, A. E. V. (1973) Proteins: A Guide to Study by Physical and Chemical Methods, 1st edn., pp. 219-224, John Wiley and Sons, New York Spector, A. R. (1967) Ph.D. Thesis, Northwestern University Layne, E. in Methods of Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 3, pp. 447454, Academic Press, New York
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37274 P U R I F I C A T I O N A N D SOME P R O P E R T I E S OF T H E P H O S P H O P R O T E I N FROM RAT INCISORS
WILLIAM T. BUTLER, WILLIAM T. HALL and WILLIAM S. RICHARDSON Institute of Dental Research, University of Alabama in Birmingham, University Station, Birmingham, Ala. 35294 (U.S.A.)
(Received August 13th, 1975)
SUMMARY The phosphoprotein of rat incisors has been purified by successive gel and ion-exchange chromatography. The product gave a single band on polyacrylamide gel electrophoresis and contained approximately 3 4 ~ phosphoserine and 3 2 ~ aspartic acid. Alkaline elimination experiments showed all the phosphate to be present as phosphoserine. Ultraviolet spectra in the presence or absence of ATP showed that the phosphoprotein did not contain a nucleotide moiety as suggested by Veis, A., Spector, A. R. and Zamoscianyk, H. ((1972) Biochim. Biophys. Acta 257, 404-413) for bovine dentin phosphoprotein.
INTRODUCTION One of the organic constituents of dentin is a polyanionic protein containing high levels of phosphate, serine and aspartate. In the unerupted bovine molar this phosphoprotein is only partially soluble and is believed to occur in two forms: insoluble, covalently bound to collagen [1] and soluble, conjugated to a nucleotide like component [2, 3]. In contrast, the rat phosphoprotein is almost completely soluble [4]. This report summarizes a procedure for purifying the rat phosphoprotein and presents evidence that the phosphoprotein from this species does not occur conjugated to a nucleotide. MATERIALS AND METHODS Extraction and purification
The phosphoprotein was extracted from rat incisors essentially as described [4]. The extract was desalted on a column of Bio-Gel P-2 (Bio-Rad, 100-200 mesh) and eluted with 0.2 M acetic acid and lyophilyzed. A 20 mg sample was dissolved in 5 ml of 0.01 M Tris-HC1 buffer (pH 7.4) and placed on a 2.5 x 60 cm column of Sephadex G-150 (Pharmacia, medium) and eluted with the dissolving buffer (Fig. l). The effluent was monitored at 230 nm with a Beckman DB-G spectrophotometer equipped with a flow cell. Fractions of 5 ml were collected and aliquots were assayed for phosphate liberated after hydrolysis
263 with 0.2 M N a O H at 37 °C for 24 h. Phosphate analyses were performed with a Technicon Auto-Analyzer [5]. Phosphate-containing fractions from the Sephadex G-150 column were pooled and directly applied to a 2.0 × 20 cm column of DEAE-cellulose (Whatman DE-32, microgranular) previously equilibrated with 0.01 M Tris-HC1 buffer (pH 7.4). It was eluted with a linear gradient from 0 to 1.0 M NaC1 over total volume of 1 liter (Fig. 2). The effluent was monitored as described in the preceding paragraph. Phosphoprotein tractions were desalted as described above and lyophilyzed.
Analysis Disc gel electrophoresis was by the method of Davis [6] with 7 ~ polyacrylamide gels. Samples of 0.2 to 0.3 mg were applied and, after runs of 45 min at 7 mA per tube, the gels were stained with Coomassie Blue [7] and destained by diffusion in 20 ~ acetic acid. For amino acid and phosphate analysis of the purified phosphoprotein, approximately 2-rag samples, previously dried in vacuo over Drierite, were hydrolyzed in constant boiling HC1 at 108 °C for 24 h. Hydrolysis was under reduced pressure in a vacuum desicator which was saturated with 6 M HC1 [8]. Both analyses were performed on aliquots of the same hydrolyzates. Amino acid analysis was with a Beckman 120C analyzer modified for single-column analysis [9] (Table I). Analyses foi phosphate were as outlined above; results were compared to standards containing 0.05 to 0.80 #mol per ml NaH2PO4" H20. The observed amount of serine measured on the amino acid analyzer arises from seryl and phosphoseryl residues which are destroyed by acid hydrolysis at different rates. Since the phosphoserine value was known from the measured phosphate and, assuming that 10 ~ of the serine [10] and 25 ~o of the phosphoserine [11] were destroyed, the amount of serine could then be calculated from the algebraic expression : Serob~ = 0.90 Ser + 0.75 Psr where Serobs represents the amount of serine measured with the amino acid analyzer. To determine if all the phosphate of the phosphoprotein were present as phosphoserine, the protein was subjected to an alkaline elimination reaction. The amount of serine lost was compared to the amount of inorganic phosphate liberated. Duplicate samples were incubated at 37 °C in 0.2 M N a O H alongside controls incubated in water. Aliquots were then taken for amino acid and phosphate analyses. Analysis for calcium was performed by Galbraith Laboratories, Inc., Knoxville, Tennessee.
Ultraviolet spectral analysis The spectra of solutions of the purified phosphoprotein and of the phosphoprotein containing ATP were recorded with a Beckman Acta III recording spectrophotometer. Solutions containing 1 mg/ml and 0.125 mg/ml of phosphoprotein in 0.05 M sodium phosphate buffer (pH 7.0) in 1 cm cuvettes were initially used. Appropriate aliquots of a 1 mg/ml solution of ATP in the same buffer were added and the spectra recorded again.
264 RESULTS
Gel chromatography of the crude phosphoprotein resulted in a single major peak eluting in the void volume of the column (Fig. 1). The phosphate closely followed this excluded peak. Subsequent chromatography of the phosphoprotein on DEAEcellulose (Fig. 2) resulted in a single major peak eluting at approximately 0.4 M NaC1. Again, the phosphate corresponded closely to the ultraviolet absorption for the major peak. The resultant phosphoprotein was judged to be pure, since a single protein band was observed when the material was subjected to polyacrylamide gel electrophoresis.
14-
o
12 ~
I~l I003
&
O.2-, 50
O0
2OO
150
250
300
EFFLUENT VOLUME, ml
Fig. 1. Gel filtration o f crude rat incisor phosphoprotein. A 20 m g sample was applied to a 2.5 × 60 c m c o l u m n o f Sephadex G-150 (medium) e m p l o y i n g 0.01 M Tris-HC1 (pH 7.4) as elution buffer. The effluent was monitored for protein at 230 n m and for phosphate after treating aliquots o f the 5-ml fractions with 0.2 M N a O H for 24 h at 37 °C. Phosphate analyses were performed with a Technicon Auto-Analyzer [5]. T 0.6
E 0 o,4
0.2
o,O~ 0.0.
O0
ioo
200
300
400
500
EFFLUENT VOLUME, ml
Fig. 2. Ion-exchange chromatography of phosphoprotein. Appropriate fractions from Sephadex G 150 (Fig. 1) were pooled and directly applied to a 2.0 × 2 0 c m c o l u m n of DEAE-cellulose ( W h a t m a n DE-32, microgranular) previously equilibrated with 0.01 M Tris-HCI (pH 7.4). Elution was with a linear gradient f r o m 0 to 1.0 M NaC1 over a total volume o f I liter. The effluent was monitored as described in the legend o f Fig. 1. Phosphoprotein fractions were desalted on a column of Bio-Gel P-2 and lyophilized.
The composition of the purified phosphoprotein (Table I) was similar to that reported for other dentinal phosphoproteins [2, 12]. Phosphoserine and aspartic acid accounted for 65 % of the residues of the protein. About 80 ~ of the weight of protein hydrolyzed in these experiments could be recovered as amino acids. Phosphoseryl residues were assumed to be in the monosodium form for these calculations. The calcium content was only 0.32 ~ 0.0l %.
265 TABLE I AMINO ACID COMPOSITION OF RAT INCISOR PHOSPHOPROTEIN Amino a c i d
Residues/thousand
Phosphoserine Aspartic acid Serine Glutamic acid Glycine Threonine Alanine Lysine Proline Histidine Arginine Half cystine Leucine Valine Isoleucine Tyrosine Phenylalanine
339.0 315.0 170.0 50.1 32.4 17.9 15.4 11.4 8.1 7.0 7.0 6.9 5.8 5.2 3.8 3.1 1.6
The amount of serine destroyed by treatment of the phosphoprotein with mild alkali corresponded closely to the amount of inorganic phosphate liberated by this procedure. For example, in one experiment the loss of serine was 2.61, and the level o f nascent phosphate was 2.67/zmol per mg of sample. Threonine levels remained constant during the treatment. In view of the conclusion that the soluble phosphoprotein from bovine molars is conjugated to a nucleotide [2, 3], we decided to examine the effect o f various amounts of ATP upon the ultraviolet spectral properties of the rat incisor phosphoprotein. The spectrum of solutions containing 1 mg/ml or 0.125 mg/ml (Fig. 3) of the phosphoprotein showed no discrete absorption peaks in the 230 to 300 nm range, but a large absorbance peak at approximately 205 nm, the n -~ zt* transition of the t0 o9
C\ F
O.8 w07 0 Z 06
~04 m '~ 0.3 02 0.1 200
220
240
260
280
300
WAVELENGTH, nm
Fig. 3. Ultraviolet spectrum of rat incisor phosphoprotein ( - - - - - - , 0.125 mg/ml) and rat incisor phosphoprotein with 5 ~ ATP ( - - - - ,0.120 mg/ml protein and 0.006 mg/ml ATP).
266 peptide groups [13]. Upon addition of ATP equal to 5 ~ of the weight of the phosphoprotein a distinct absorbance peak at 260 nm was observed (Fig. 3). A slight increase in the 205 nm peak was also observed under these conditions. When the solution contained equal weights of ATP and phosphoprotein (0.060 mg/ml), both the 260 and 205 nm peaks were quite large. Control experiments and calculations utilizing the extinction coefficient of ATP showed that the presence of the phosphoprotein did not alter the level of the 260 nm absorbance of ATP. DISCUSSION Veis et al. [2] have reported that the phosphoprotein from bovine dentin is conjugated to a nonprotein moiety believed to be a "purine, an adenine derivative" [3]. The conclusion that a nonprotein substituent accounts for about 5 0 ~ of the weight of the phosphoprotein was based on the inability to account for the weight of the preparation as amino acids and the fact that the 260 nm absorbance of the protein was greater than that at 280 nm [2]. In addition, the total phosphorus found by elemental analysis was greater than could be accounted for as phosphoserine with the methods employed. In contrast, about 80 ~ of the weight of the phosphoprotein from rat incisors could be accounted for as amino acids. While the remainder of the weight was neither carbohydrate [12] nor calcium, it could have been tightly bound water or unknown metals. The alkaline elimination experiments clearly demonstrated that all the phosphate could be accounted for as phosphoserine. The ultraviolet spectral studies (Fig. 3) showed that the addition of A T P gave a discrete 260 nm absorbance peak not seen in the solutions containing only the phosphoprotein. Thus there is no evidence that the rat phosphoprotein contained a nucleotide, though the spectrum of the phosphoprotein would have ieadily revealed the presence of less than 5 ~ by weight of nucleotide (Fig. 3). If 50 ~o of the weight of the protein did indeed consist of a nucleotide, a spectrum with a strong 260 nm absorbance should have been obtained. Veis et al. [2] and Spector [14] have reported spectral data for the bovine phosphoprotein. The ultraviolet spectrum [14] displayed only slight absorbances at 260 nm and 280 nm and a strong absorbance below 230 nm. We evaluated this spectrum for nucleotide content, using the extinction coefficient of ATP at 260 nm, and for protein content reflected by the 230 nm absorbance (compared to that of a known amount of rat incisor phosphoprotein with a remarkably similaI amino acid content). We conclude that the bovine phosphoprotein could contain no more than 2 ~ by weight of nucleotide. In addition, the reported 260 nm to 280 nm ratio of the bovine phosphoprotein was approximately 2.0 [2, 14], indicating the possibility of high nucleotide content. However, Layne [15] reviewed the use of relative absorbances at 260 nm and 280 nm for the determination of pl otein in the presence of nucleotides. Only proteins with distinct 280 nm absorbances were candidates for the technique and, even then, the determinations had the potential for considerable error. We, thelefore, strongly suggest that, sir, ce the level of aromatic amino acids in the bovine phosphoprotein [2, 14] is extremely low and the 280 nm absorbance [14] is correspordingly weak, that a ratio of 260 nm to 280 nm is not a reliable indicator for the presence of a nucleotide.
267 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Veis, A. and Perry, A. (1967) Biochemistry 6, 2409-2416 Veis, A., Spector, A. R. and Zamoscianyk, H. (1972) Biochim. Biophys. Acta 257, 404-413 Sharkey, M. A. and Veis, A. (1974) J. Dental Res. 53 (Special Issue), 57-63 Butler, W. T., Finch, J. E. and DeSteno, C. V. (1972) Biochim. Biophys. Acta 257, 167-171 Technicon Auto-Analyzer Methodology (1968) Technicon Corp., Tarrytown, N.Y. Davis, B. T. (1964) Ann. N.Y. Acad. Sci. 121, 404-427 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 Dreyer, W. J. and Bynum, E. (1967) in Methods of Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. 11, pp. 32-39, Academic Press, New York Miller, E. J. and Piez, K. A. (1966) Anal. Biochem. 16, 320-326 Downs, F. and Pigman, W. (1970) Int. J. Protein Res. 2, 27-36 Allerton, S. E. and Perlman, G. E. (1965) J. Biol. Chem. 240, 3892-3898 Butler, W. T. (1972) in The Comparative Molecular Biology of Extracellular Matrices (Slavkin, H. C., ed.), pp. 255-259, Academic Press, New York Haschemeyer, R. H. and Haschemeyer, A. E. V. (1973) Proteins: A Guide to Study by Physical and Chemical Methods, 1st edn., pp. 219-224, John Wiley and Sons, New York Spector, A. R. (1967) Ph.D. Thesis, Northwestern University Layne, E. in Methods of Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 3, pp. 447454, Academic Press, New York