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The conversion of ovalbumin into plakalbumin, as followed in the pH-stat
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 70-77 (1966)
The Conversion of Ovalburnin into Plakalbumin, as Followed in the pa-stat M. Ottesen From the Carlsberg Laboratory, Copenhagen, Denmark Received June 4, 1956
INTRODUCTION In a series of papers it has been shown that subtilisin, the proteinase obtained from a strain of Bacillus subtilis, is able to attack native ovalbumin, transforming it into another native protein, plakalbumin (l-3). In most of the previous studies this reaction was carried out at pH 6.4. The results indicated that the ovalbumin-plakalbumin transformation was invariably accompanied by liberation of peptide material, chiefly alanylalanine; a hexapeptide (ala-gly-val-asp-ala-ala) and a tetrapeptide (ala-gly-val-asp) were also found. In an investigation of the action of carboxypeptidase on ovalbumin, Steinberg (4) found that,, although ovalbumin is resistant to carboxypeptidase, addition of trace amounts of subtilisin at pH 7.8 makes ovalbumin susceptible to attack by carboxypeptidase, C-terminal alanine being liberated. The amount of subtilisin used was insufhcient to cause appreciable peptide liberation. The experiments therefore suggest that the initial attack of subtilisin upon ovalbumin consists in the opening of a single peptide bond in a closed peptide chain of the ovalbumin molecule, giving rise to the C-terminal alanine. In the present paper, it has been attempted to verify this picture by following the opening of peptide bonds during the ovalbumin-plakalbumin transformation at pH 8. MATERIALS The crystalline ovalbumin used was prepared according to SIdrensen and Heyrup (6). It w&s freed of salt by dialysis for 4-5 weeks against distilled water. The solution was filtered, brought to the desired pH with 0.1 N NaOH, diluted to the appropriate concentration, and stored at ea. 2°C. under toluene until it was used. The enzyme employed was four-times crystallized subtilisin, prepared as previously described (6). The stock solution contained 1 mg. enzyme/ml. It was stored in 0.5-ml. portions in the frozen state. 70
OVALBUMIN INTO PLAKALBUMIN
71
METHODS The methods ordinarily used to follow the opening of peptide bonds are titration with base in alcoholic medium or with acid in acetone. In the case of ovalbumin, these methods are not very useful, since they cause precipitation of the protein, resulting in lowered accuracy. However, it is possible under some conditions to follow the opening of peptide bonds in an aqueous medium by continuous titration with base at a constant PH. The principle of this method is as follows: Hydrolysis of a peptide bond will release hydrogen ions into the solution, provided the pH is high enough so that nonionized amino groups will be formed. The amount of hydrogen ions released per mole peptide bond cleaved is determined by the well-known equation
[NIL- RI pH = pK + ‘pg [N&+-R] This procedure was first utilized by Waley and Watson (7). In the Carlsberg laboratory the method was refined by the introduction of the pH-stat, a device which automatically keeps the pH constant by addition of the required amount of base. A description of this apparatus has been given elsewhere @).I In the present case the proteolysis was only around one peptide bond per mole protein. This called for precautions to ensure maximum stability of the system. Not only was the reaction mixture kept at constant temperature in a water bath, but the whole setup was placed in a constant-temperature room, having a temperature 1°C. below that of the water thermostat, which was 30°C. For each titration was used 10 ml. of a 5% protein solution; the base was 0.1 N NaOH. Carbon dioxide was flushed from the reaction vessel by a slow stream of nitrogen. When the desired pH had been reached, from 3 to 50 pl. of the enzyme solution was added to the substrate. The pH-stat connected to the mechanical recorder regis-, tered the alkali uptake as a function of time. In experiments lasting a couple of hours the curves were generally reproducible within co. 0.05 mole of alkali per mole protein. The increase in solubility accompanying the transformation of ovalbumin into plakalbumin was followed by titration of samples with ammonium sulfate solution to the appearance of a turbidity as previously described (1). The crystallizations and the nonprotein nitrogen determinations were performed in the usual manner (1).
RESULTS The Alkali
Uptake Measured in the pH-stat
In a series of experiments the reaction between ovalbumin and subtilisin at pH 8 and 30°C. was followed by means of the PI-I-stat. Figure 1 shows some typical curves for the base uptake as a function of time at different enzyme concentrations. All the curves show a relatively fast 1 Jacobsen, C. F., Leonis, J., Linderstrem-Lang, of Biochemical Analysis,” in preparation.
K., and Ottesen, M., “Methods
72
M.
O’ITESEN
FIG. 1. The uptake of base registered with the pH-stat during the reaction between ovalbumin and different concentrations of subtilisin, at pH 8 and 30°C. Enzyme concentrations are given as micrograms of enzyme per gram of protein. The abscissa is reaction time in hours. The ordinate is uptake of base measured as mole of base per mole of protein.
initial reaction, in the following called the primary reaction, followed by a slower reaction proceeding at a practically constant rate: the secondary reaction. If the rectilinear curve representing the secondary reaction is extrapolated back to zero time, its intersection with the axis of ordinates determines the extent of the primary reaction. It will be seen that the uptake due to the primary reaction is independent of enzyme concentration within the range 30-100 pg. enzyme/g. protein, and that it corresponds to cu. 0.9 mole base per mole protein. This corresponds to the amount expected for the opening of one peptide bond in the protein molecule. A few analogous experiments have been performed at other pH values, viz., pH 6.4-7.5-8.5, and the over-all base uptake varied in the way expected for the opening of peptide bonds. In contrast, the experiments performed with enzyme concentrations below cu. 30 pg./g. protein showed a primary reaction of less than 0.9 mole, decreasing with decreasing enzyme concentrations. The secondary reaction was absent, since the corresponding part of the curve was parallel to the blank curve obtained with substrate alone. Hence it
OVALBUMIN
INTO
PLAKALBUMIN
73
appears that the enzyme is inactivated before the primary reaction is completed. Experiments in which small portions of enzyme were added at different stages of the reaction showed that the inactivation must be due to stoichiometric combination of the enzyme with some inhibitor, not to a general instability of the enzyme in the dilute solutions. The rates of the secondary reaction at higher enzyme concentrations are consistent with this picture, but we have as yet no information about the nature of the inhibitor. The Correlation between Alkali Uptake and Increase in Solubility Next it was attempted to correlate the base uptake as measured in the pH-stat with the increase in solubility characteristic of the transformation of ovalbumin into plakalbumin. During pH-stat experiments, samples of the reaction mixture were taken out at appropriate time intervals, and the solubility was determined by titration with ammonium sulfate (AMS) solution to the turbidity point. Some curves correlating alkali uptake with solubility increase are seen in Fig. 2. Under the experimental conditions used, ovalbumin samples are titrated with 2.6-2.7 ml. AMS, whereas plakalbumin samples require 3.3-3.4 ml. AMS. It will be seen from the figure that the primary reaction is accompanied
MO/es Off- added/mote
of protein
2. The correlation between the uptake of base registered in the pH-stat and the increase in solubility during the reaction between ovalbumin and subtilisin at 13, 30, and 50 pg. enzyme/g. protein. FIG.
74
M.
O’ITESEN
by an increase in solubility. However, the curve levels off at cu. 3.0-3.1 ml. AMS after completion of the primary reaction, and only in later stages does the solubility increase to the level characteristic of plakalbumin. When the enzyme concentration is insufficient to complete the primary reaction, it is also unable to increase the solubility to the intermediary level mentioned. Correlation between Alkali
Uptake and Crystal Form
The reaction between ovalbumin and subtilisin was followed as usual in the pa-stat, and after an appropriate reaction time the protein solution was brought to pH 5.5, and crystallization with AMS solution was attempted. It turned out that reaction mixtures in which the primary reaction had been completed would never crystallize in the needles characteristic of ovalbumin. Furthermore, if the primary reaction was only partially completed, fractional crystallization showed that the disappearance of the needle crystals was approximately proportional to the extent of the primary reaction, Ovalbumin, as well as reaction mixtures in which the transformation to plakalbumin had been completed, would crystallize within a few hours. In contrast, reaction mixtures rich in the intermediary protein would only crystallize after a couple of days’ standing; they then formed crystals similar to the plakalbumin crystals. The slow crystallization of this intermediary compound might possibly indicate that it in itself is unable to crystallize. Only after some further transformation will it crystallize as plakalbumin. Correlation between Alkali Uptake and Liberation of Nonprotein Nitrogen The opening of peptide bonds causes alkali uptake as measured with the pH-stat irrespective of whether the opening leads to the actual splitting off of a peptide or not. Since peptides normally are released into solution in the reaction between subtilisin and ovalbumin, we have investigated to what extent the measured base consumption might arise from liberation of peptides. The digestions. were carried out as usual in the pa-stat. When a predetermined quantity of base had been consumed, digestion was terminated by acidification, and the amount of nonprotein nitrogen (NPN) formed during the reaction was determined. Figure 3 shows the correlation between base uptake and release of NPN. The interpretation of these results is complicated by the fact that the correct amount of base used to titrate the liberated peptides can only
OVALBUMIN
INTO
moles FIG. 3. The correlation
between the uptake of base registered in the pH-stat and subtilisin. The values for the base blank value obtained from the substrate
75
PLAKALBUMIN
OF NPN
release of nonprotein nitrogen and the during the reaction between ovalbumin uptake have been corrected for the low alone.
be calculated if the exact amount of and the pK value for each individual peptide are known. As yet we do not have such information, but we know that the NPN fraction consists of peptides containing the amino acids alanine, glycine, valine, aspartic acid, and glutamic acid. Using the pK values listed by Cohn and Edsall (9) for peptides composed of such amino acids, it is possible to calculate the base uptake arising from the liberation of one mole of any such peptide at pH 8 at 30°C. from an open peptide chain. It turns out to approach 0.25 mole of base per mole peptide nitrogen for dipeptides, and to be lower for larger peptides. The line corresponding to this maximal base uptake, 0.25 mole per mole NPN, is shown in Fig. 3, and it is seen that only a fraction of the measured base uptake can be due to the liberation of peptides. Consequently the rest arises from the opening of peptide bonds in the interior of the ovalbumin molecule.
DISCUSSION The experiments described in the preceding sections demonstrate that the transformation of ovalbumin to plakalbumin at pH 8 consists of at least two steps. The first step has been investigated in detail, and it was found that during this process the protein loses its ability to crystallize in needles, the solubility is somewhat increased, and in the pa-stat an alkali uptake corresponding to cu. 0.9 mole per mole protein is measured. This value was found by linear extrapolation, but since part of the en-
76
M. O'ITESEN
zyme was found to be inactivated during the primary reaction it most likely represents a maximum value. The base uptake due to the opening of the first peptide bond in the ovalbumin molecule can also be estimated from Fig. 3. The base uptake arising from the liberation of alanylalanine is 0.17 mole per mole nitrogen, and if we assume this reaction to be the dominating one in the initial part, we find that the base uptake arising from the opening of peptide bonds in the interior of the ovalbumin molecule is cu. 0.5 mole base per mole protein, corresponding to the opening of a single peptide bond, if the amino group involved has the pK value co. 8. Thus the experiments confirm the theory proposed by Steinberg (4) that the initial attack of subtilisin upon ovalbumin consists in the opening of a single peptide bond in a closed peptide chain, leading to the formation of an open, intermediary type of protein, which in turn is further degraded to plakalbumin. Reaction mixtures rich in this intermediary protein crystallize only with difficulty, and when they do so, they form plates. It is unclear whether this intermediary protein in itself crystallizes in plates or whether it has to be transformed into another protein before it is able to form the plate crystals. Ovalbumin is known to be devoid of N-terminal amino groups, but since the intermediary protein is formed by the opening of a peptide bond, both this form and the resulting plakalbumin should have a free N-terminal amino group. Attempts to confirm the existence of such a group and to determine its nature are being made using the technique of Sanger (10). DNP-serine has been isolated, but so far in unsatisfactorily low yields. If further experiments confirm that serine is the N-terminal amino acid in plakalbumin, the bond first opened in ovalbumin by subtilisin would be one from alanine to serine. ACKNOWLEDGMENTS The author wishes to express his deep appreciation to Professor K. Linderstr#m-Lang for stimulation and inspiration, not only in this investigation but throughout our association. He also wishes to thank Mrs. J. Hjo Taplov for expert technical assistance. SUMMARY
The limited proteolysis accompanying the transformation of ovalbumin into plakalbumin has been measured using the pa-stat. The reaction consists of at least two steps, the first step being the opening of a single peptide bond in a closed chain in the ovalbumin molecule,
OVALBUMIN INTO PLAKALBUMIN
77
leading to the formation of an open intermediary protein form which has lost the ability of crystallizing in needles. In the further reaction this form is converted into plakalbumin. REFERENCES 1. LINDERSTR$M-LANG, K., AND OTTESEN, M., Compt. rend. trav. lab. Carlsberg. SBr. chim. 26, 403 (1949). 2. OTTESEN, M., AND VILLEE, C., Compt. rend. trav. lab. Curlsberg. SBr. chim. 27, 421 (1951). 3. OTTESEN, M., AND WOLLENBERGER, A., Compt. rend. trav. lab. Carlsberg. St%. chim. 26, 463 (1953). 4. STEINBERQ, D., Compt. rend. trav. lab. Carlsberg. Sbr. chim. 29, 176 (1954). 5. S$RENSEN, S. P. L., AND H$YRUP, M., Compt. rend. trav. lab. Carlsberg. SBr. chim. 12, 12 (1917). 6. GBNTELBERG, A., AND OTTESEN, M., Compt. rend. trav. lab. Carlsberg. SCr. chim. 29, 36 (1954). 7. WALEY, S. G., AND WATSON, J., Biochem. J. 66, 328 (1953). 8. JACOBSEN, C. F., AND LBONIS, J., Compt. rend. trav. lab. Carlsberg. Dr. chim. 27, 333 (1951). 9. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides,” Reinhold, New York, 1943. 10. SANGER, F. Biochem. J. 39, 507 (1945).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 70-77 (1966)
The Conversion of Ovalburnin into Plakalbumin, as Followed in the pa-stat M. Ottesen From the Carlsberg Laboratory, Copenhagen, Denmark Received June 4, 1956
INTRODUCTION In a series of papers it has been shown that subtilisin, the proteinase obtained from a strain of Bacillus subtilis, is able to attack native ovalbumin, transforming it into another native protein, plakalbumin (l-3). In most of the previous studies this reaction was carried out at pH 6.4. The results indicated that the ovalbumin-plakalbumin transformation was invariably accompanied by liberation of peptide material, chiefly alanylalanine; a hexapeptide (ala-gly-val-asp-ala-ala) and a tetrapeptide (ala-gly-val-asp) were also found. In an investigation of the action of carboxypeptidase on ovalbumin, Steinberg (4) found that,, although ovalbumin is resistant to carboxypeptidase, addition of trace amounts of subtilisin at pH 7.8 makes ovalbumin susceptible to attack by carboxypeptidase, C-terminal alanine being liberated. The amount of subtilisin used was insufhcient to cause appreciable peptide liberation. The experiments therefore suggest that the initial attack of subtilisin upon ovalbumin consists in the opening of a single peptide bond in a closed peptide chain of the ovalbumin molecule, giving rise to the C-terminal alanine. In the present paper, it has been attempted to verify this picture by following the opening of peptide bonds during the ovalbumin-plakalbumin transformation at pH 8. MATERIALS The crystalline ovalbumin used was prepared according to SIdrensen and Heyrup (6). It w&s freed of salt by dialysis for 4-5 weeks against distilled water. The solution was filtered, brought to the desired pH with 0.1 N NaOH, diluted to the appropriate concentration, and stored at ea. 2°C. under toluene until it was used. The enzyme employed was four-times crystallized subtilisin, prepared as previously described (6). The stock solution contained 1 mg. enzyme/ml. It was stored in 0.5-ml. portions in the frozen state. 70
OVALBUMIN INTO PLAKALBUMIN
71
METHODS The methods ordinarily used to follow the opening of peptide bonds are titration with base in alcoholic medium or with acid in acetone. In the case of ovalbumin, these methods are not very useful, since they cause precipitation of the protein, resulting in lowered accuracy. However, it is possible under some conditions to follow the opening of peptide bonds in an aqueous medium by continuous titration with base at a constant PH. The principle of this method is as follows: Hydrolysis of a peptide bond will release hydrogen ions into the solution, provided the pH is high enough so that nonionized amino groups will be formed. The amount of hydrogen ions released per mole peptide bond cleaved is determined by the well-known equation
[NIL- RI pH = pK + ‘pg [N&+-R] This procedure was first utilized by Waley and Watson (7). In the Carlsberg laboratory the method was refined by the introduction of the pH-stat, a device which automatically keeps the pH constant by addition of the required amount of base. A description of this apparatus has been given elsewhere @).I In the present case the proteolysis was only around one peptide bond per mole protein. This called for precautions to ensure maximum stability of the system. Not only was the reaction mixture kept at constant temperature in a water bath, but the whole setup was placed in a constant-temperature room, having a temperature 1°C. below that of the water thermostat, which was 30°C. For each titration was used 10 ml. of a 5% protein solution; the base was 0.1 N NaOH. Carbon dioxide was flushed from the reaction vessel by a slow stream of nitrogen. When the desired pH had been reached, from 3 to 50 pl. of the enzyme solution was added to the substrate. The pH-stat connected to the mechanical recorder regis-, tered the alkali uptake as a function of time. In experiments lasting a couple of hours the curves were generally reproducible within co. 0.05 mole of alkali per mole protein. The increase in solubility accompanying the transformation of ovalbumin into plakalbumin was followed by titration of samples with ammonium sulfate solution to the appearance of a turbidity as previously described (1). The crystallizations and the nonprotein nitrogen determinations were performed in the usual manner (1).
RESULTS The Alkali
Uptake Measured in the pH-stat
In a series of experiments the reaction between ovalbumin and subtilisin at pH 8 and 30°C. was followed by means of the PI-I-stat. Figure 1 shows some typical curves for the base uptake as a function of time at different enzyme concentrations. All the curves show a relatively fast 1 Jacobsen, C. F., Leonis, J., Linderstrem-Lang, of Biochemical Analysis,” in preparation.
K., and Ottesen, M., “Methods
72
M.
O’ITESEN
FIG. 1. The uptake of base registered with the pH-stat during the reaction between ovalbumin and different concentrations of subtilisin, at pH 8 and 30°C. Enzyme concentrations are given as micrograms of enzyme per gram of protein. The abscissa is reaction time in hours. The ordinate is uptake of base measured as mole of base per mole of protein.
initial reaction, in the following called the primary reaction, followed by a slower reaction proceeding at a practically constant rate: the secondary reaction. If the rectilinear curve representing the secondary reaction is extrapolated back to zero time, its intersection with the axis of ordinates determines the extent of the primary reaction. It will be seen that the uptake due to the primary reaction is independent of enzyme concentration within the range 30-100 pg. enzyme/g. protein, and that it corresponds to cu. 0.9 mole base per mole protein. This corresponds to the amount expected for the opening of one peptide bond in the protein molecule. A few analogous experiments have been performed at other pH values, viz., pH 6.4-7.5-8.5, and the over-all base uptake varied in the way expected for the opening of peptide bonds. In contrast, the experiments performed with enzyme concentrations below cu. 30 pg./g. protein showed a primary reaction of less than 0.9 mole, decreasing with decreasing enzyme concentrations. The secondary reaction was absent, since the corresponding part of the curve was parallel to the blank curve obtained with substrate alone. Hence it
OVALBUMIN
INTO
PLAKALBUMIN
73
appears that the enzyme is inactivated before the primary reaction is completed. Experiments in which small portions of enzyme were added at different stages of the reaction showed that the inactivation must be due to stoichiometric combination of the enzyme with some inhibitor, not to a general instability of the enzyme in the dilute solutions. The rates of the secondary reaction at higher enzyme concentrations are consistent with this picture, but we have as yet no information about the nature of the inhibitor. The Correlation between Alkali Uptake and Increase in Solubility Next it was attempted to correlate the base uptake as measured in the pH-stat with the increase in solubility characteristic of the transformation of ovalbumin into plakalbumin. During pH-stat experiments, samples of the reaction mixture were taken out at appropriate time intervals, and the solubility was determined by titration with ammonium sulfate (AMS) solution to the turbidity point. Some curves correlating alkali uptake with solubility increase are seen in Fig. 2. Under the experimental conditions used, ovalbumin samples are titrated with 2.6-2.7 ml. AMS, whereas plakalbumin samples require 3.3-3.4 ml. AMS. It will be seen from the figure that the primary reaction is accompanied
MO/es Off- added/mote
of protein
2. The correlation between the uptake of base registered in the pH-stat and the increase in solubility during the reaction between ovalbumin and subtilisin at 13, 30, and 50 pg. enzyme/g. protein. FIG.
74
M.
O’ITESEN
by an increase in solubility. However, the curve levels off at cu. 3.0-3.1 ml. AMS after completion of the primary reaction, and only in later stages does the solubility increase to the level characteristic of plakalbumin. When the enzyme concentration is insufficient to complete the primary reaction, it is also unable to increase the solubility to the intermediary level mentioned. Correlation between Alkali
Uptake and Crystal Form
The reaction between ovalbumin and subtilisin was followed as usual in the pa-stat, and after an appropriate reaction time the protein solution was brought to pH 5.5, and crystallization with AMS solution was attempted. It turned out that reaction mixtures in which the primary reaction had been completed would never crystallize in the needles characteristic of ovalbumin. Furthermore, if the primary reaction was only partially completed, fractional crystallization showed that the disappearance of the needle crystals was approximately proportional to the extent of the primary reaction, Ovalbumin, as well as reaction mixtures in which the transformation to plakalbumin had been completed, would crystallize within a few hours. In contrast, reaction mixtures rich in the intermediary protein would only crystallize after a couple of days’ standing; they then formed crystals similar to the plakalbumin crystals. The slow crystallization of this intermediary compound might possibly indicate that it in itself is unable to crystallize. Only after some further transformation will it crystallize as plakalbumin. Correlation between Alkali Uptake and Liberation of Nonprotein Nitrogen The opening of peptide bonds causes alkali uptake as measured with the pH-stat irrespective of whether the opening leads to the actual splitting off of a peptide or not. Since peptides normally are released into solution in the reaction between subtilisin and ovalbumin, we have investigated to what extent the measured base consumption might arise from liberation of peptides. The digestions. were carried out as usual in the pa-stat. When a predetermined quantity of base had been consumed, digestion was terminated by acidification, and the amount of nonprotein nitrogen (NPN) formed during the reaction was determined. Figure 3 shows the correlation between base uptake and release of NPN. The interpretation of these results is complicated by the fact that the correct amount of base used to titrate the liberated peptides can only
OVALBUMIN
INTO
moles FIG. 3. The correlation
between the uptake of base registered in the pH-stat and subtilisin. The values for the base blank value obtained from the substrate
75
PLAKALBUMIN
OF NPN
release of nonprotein nitrogen and the during the reaction between ovalbumin uptake have been corrected for the low alone.
be calculated if the exact amount of and the pK value for each individual peptide are known. As yet we do not have such information, but we know that the NPN fraction consists of peptides containing the amino acids alanine, glycine, valine, aspartic acid, and glutamic acid. Using the pK values listed by Cohn and Edsall (9) for peptides composed of such amino acids, it is possible to calculate the base uptake arising from the liberation of one mole of any such peptide at pH 8 at 30°C. from an open peptide chain. It turns out to approach 0.25 mole of base per mole peptide nitrogen for dipeptides, and to be lower for larger peptides. The line corresponding to this maximal base uptake, 0.25 mole per mole NPN, is shown in Fig. 3, and it is seen that only a fraction of the measured base uptake can be due to the liberation of peptides. Consequently the rest arises from the opening of peptide bonds in the interior of the ovalbumin molecule.
DISCUSSION The experiments described in the preceding sections demonstrate that the transformation of ovalbumin to plakalbumin at pH 8 consists of at least two steps. The first step has been investigated in detail, and it was found that during this process the protein loses its ability to crystallize in needles, the solubility is somewhat increased, and in the pa-stat an alkali uptake corresponding to cu. 0.9 mole per mole protein is measured. This value was found by linear extrapolation, but since part of the en-
76
M. O'ITESEN
zyme was found to be inactivated during the primary reaction it most likely represents a maximum value. The base uptake due to the opening of the first peptide bond in the ovalbumin molecule can also be estimated from Fig. 3. The base uptake arising from the liberation of alanylalanine is 0.17 mole per mole nitrogen, and if we assume this reaction to be the dominating one in the initial part, we find that the base uptake arising from the opening of peptide bonds in the interior of the ovalbumin molecule is cu. 0.5 mole base per mole protein, corresponding to the opening of a single peptide bond, if the amino group involved has the pK value co. 8. Thus the experiments confirm the theory proposed by Steinberg (4) that the initial attack of subtilisin upon ovalbumin consists in the opening of a single peptide bond in a closed peptide chain, leading to the formation of an open, intermediary type of protein, which in turn is further degraded to plakalbumin. Reaction mixtures rich in this intermediary protein crystallize only with difficulty, and when they do so, they form plates. It is unclear whether this intermediary protein in itself crystallizes in plates or whether it has to be transformed into another protein before it is able to form the plate crystals. Ovalbumin is known to be devoid of N-terminal amino groups, but since the intermediary protein is formed by the opening of a peptide bond, both this form and the resulting plakalbumin should have a free N-terminal amino group. Attempts to confirm the existence of such a group and to determine its nature are being made using the technique of Sanger (10). DNP-serine has been isolated, but so far in unsatisfactorily low yields. If further experiments confirm that serine is the N-terminal amino acid in plakalbumin, the bond first opened in ovalbumin by subtilisin would be one from alanine to serine. ACKNOWLEDGMENTS The author wishes to express his deep appreciation to Professor K. Linderstr#m-Lang for stimulation and inspiration, not only in this investigation but throughout our association. He also wishes to thank Mrs. J. Hjo Taplov for expert technical assistance. SUMMARY
The limited proteolysis accompanying the transformation of ovalbumin into plakalbumin has been measured using the pa-stat. The reaction consists of at least two steps, the first step being the opening of a single peptide bond in a closed chain in the ovalbumin molecule,
OVALBUMIN INTO PLAKALBUMIN
77
leading to the formation of an open intermediary protein form which has lost the ability of crystallizing in needles. In the further reaction this form is converted into plakalbumin. REFERENCES 1. LINDERSTR$M-LANG, K., AND OTTESEN, M., Compt. rend. trav. lab. Carlsberg. SBr. chim. 26, 403 (1949). 2. OTTESEN, M., AND VILLEE, C., Compt. rend. trav. lab. Curlsberg. SBr. chim. 27, 421 (1951). 3. OTTESEN, M., AND WOLLENBERGER, A., Compt. rend. trav. lab. Carlsberg. St%. chim. 26, 463 (1953). 4. STEINBERQ, D., Compt. rend. trav. lab. Carlsberg. Sbr. chim. 29, 176 (1954). 5. S$RENSEN, S. P. L., AND H$YRUP, M., Compt. rend. trav. lab. Carlsberg. SBr. chim. 12, 12 (1917). 6. GBNTELBERG, A., AND OTTESEN, M., Compt. rend. trav. lab. Carlsberg. SCr. chim. 29, 36 (1954). 7. WALEY, S. G., AND WATSON, J., Biochem. J. 66, 328 (1953). 8. JACOBSEN, C. F., AND LBONIS, J., Compt. rend. trav. lab. Carlsberg. Dr. chim. 27, 333 (1951). 9. COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides,” Reinhold, New York, 1943. 10. SANGER, F. Biochem. J. 39, 507 (1945).