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On the tryptophan determination in lysozyme and its photoxidation products
ANALYTICAL
24, 80-89 (1968)
BIOCHEMISTRY
On the Tryptophan
Determination Photoxidation
V. KH. LAPUK, The
in Lysozyme
and
Its
Products
L. A. CHISTYAKOVA,
N. A. KRAVCHENKO
AND
Laboratory of Protein Chemi&ry, Institute USSR Academy of Sciences, Leninski prospekt
of
Organic
47, Moscow
Chemistry,
B-334, USSR
Received July 28, 1967 All the existing techniques of tryptophan determination in proteins may be classified into two large groups: (1) determination of tryptophan after its isolation from protein by means of chemical or enzymic hydrolysis and (2) direct determination without preliminary isolation. We applied to lysozyme photoxidation products techniques described in the literature-mild acid hydrolysis (1)) hydrolysis by pronase (2), and direct spectrophotometric method (3). These techniques were tested on native lysozyme as well as on the previously isolated products of its photoxidation by visible light (4). The tests were particularly important in this case as photoxidation was carried out to decompose tryptophan. Tryptophan photoxidation is known to be very complicated, with a gradual indole ring degradation (5). The effect of the modification of the major lysozyme chromophores on lysozyme UV spectrum, as well as the stability of the photoxidized tryptophan residues during analysis, have remained obscure. According to Ferrini (6), the products of histidine photoxidation in lysozyme change back into histidine during the usual acid hydrolysis. This fact led to a very cautious estimation of the results of the analysis of tryptophan photoxidation products. The three techniques mentioned above were chosen as the simplest, since tryptophan is not destroyed. METHODS Lysozyme was obtained by direct crystallization according to Alderton and Fevold (7), with subsequent desalting on Sephadex G-25, and by lyophilization from acetic acid (8). The products of lysozyme photoxidation were obtained by the technique described above (4) ; they are denoted by symbols X, Y, and Z, the residual activity on Micrococcus lysode-ikticus (9) being 73, 33, and 12%, respectively. Pronase was prepared in our laboratory, its activity being 0.116 X 10 1 proteinase units per milligram (10). 80
Hydrolysis
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
of lysozyme
by repeatedly
distilled
hydrochloric
81
acid (1).
5 mg of lysozyme was dissolved in 5 ml of 5-fold distilled HCI (5.7 N) . The solution was then poured into a tube, the air evacuated, and hydrolysis allowed to proceed for 15 hr at 85-90”. After drying in vacua over NaOH, the substance was analyzed by an amino acid analyzer (Czechoslovakia) . Hydrolysis
of lysoxyme
and the products
of its photoxidation
by pro-
nase (2). 44.5
mg of protein was dissolved in 3.5-4 ml of 0.2 M solution of (CH,COO)&a (ll), at pH set to 28.3, and 0.1 ml of 2.5 mg pronase solution in 1 ml of the same solvent was added. This mixture was then frozen in a Pyrex tube at -7O*, the air evacuated, and the mixture defrosted, refrozen, re-evacuated, and incubated for 2 weeks at 37-38“. The hydrolyzate was filtered and placed on a column with Amberlite CG-120, H+-form (1 X 3 cm) ; the column was washed with water until a neutral reaction was obtained and eluted by 30 ml 9N NH,OH. The eluate was dried in vacua over H,SO,. The hydrolyzate was analyzed by the amino acid analyzer. Since all the three products of photoxidation of lysozyme dissolved incompletely, the calculation of amino acid analyses on a sample was rather difficult. Therefore the calculation was made on 6 lysine residues for basic amino acids (tryptophan, lysine, histidine, arginine) and on 3 tyrosine residues (the real amount of these residues in lysozyme) for the remaining amino acids. Spectrophotometric determination of tryptophan in lysozyme and .in the products of lysozyme photoxidation by a modified technique of Bencze and Schmid (3). A protein solution of specific concentration
(within the range of 0.25-0.3 mg/ml) was prepared in 0.1 N NaOH and allowed to stand for 24 hr at 20’. After that, OD,,, and OD,,, were determined. The two points obtained were connected by a straight line in the coordinate system given in Figure 1. Here the optimum scale for calculation was as follows: ordinate, 25 mm = 0.1 OD, abscissa, 5 mm = 1 mp. Then the value s = (AOD/Ax) X lo3 Ohm was calculated. In accordance with the data in the literature (3) the dependence plot of S on R was drawn (Fig. 2)) where R is the molar tyrosine: tryptophan ratio. From Figure 2 and the calculated value S, the value R for a given protein sample, i.e., the tyrosine:tryptophan ratio, was found, either as an integer or a fractional number. Considering that tyrosine was not
82
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENEO
modified in our case, the number of tryptophan residues was determined assuming the number of tyrosine residues to be 3. RESULTS AND DISCUSSION
The routine acid hydrolysis of protein (6 N HCl, 105-llO’,’ 24 hr) is known to be unsuitable for the determination of tryptophan, the latter being fully destroyed in the course of hydrolysis (12). However in lysozyme hydrolysis with air thoroughly removed and HCl distilled in glass, the tryptophan peak appeared in many cases, its amount being not more than 3 residues. It is of interest that in these cases an unidentified peak was always present before the tryptophan peak, the ratio of their
FIG. 1. Tryptophan determination by spectrophotometric scale: 0.1 OD = 25 mm, 1 ma = 5 mm.
technique. Optimum
quantities being approximately 1:3. This suggested that the extra peak corresponded to some product of tryptophan destruction, slightly more acidic than tryptophan itself, and that it was possible to find conditions of acid hydrolysis under which tryptophan was not destroyed. This was confirmed by the data of Monier and Jutisz (1). They hydrolyzed lysozyme by repeatedly distilled hydrochloric acid at 85-90” without destroying tryptophan. We used the Monier and Jutisz technique and the results are given in Table 1. Table 1 shows that the figures on tryptophan are too low and diversified. Moreover, the extra peak presumably corresponding to the incompletely hydrolyzed peptides appears on the curves of amino acid analysis of the hydrolyzates between the lysine peak and that of histidine. All these facts indicate that the hydrolysis is not complete. The hydrolyzates obtained
are of dark,
almost
black,
color and have a gumlike
consist-
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
83
I5
i0
5
FIO. 2. Tyrosine:tryptophan relationship determination on plot drawn in accordance with data of Bencse and Schmid (3).
ency. The technique is therefore not suitable for tryptophan determination. The direct spectrophotometric method of Bencze and Schmid (3) for tryptophan (and tyrosine) determination was attempted and the results obtained for native lysozyme and the products of its photoxidation are given in Table 2. It follows from Table 2 that values of S and R close to the calculated ones for native lysozyme can be obtained only after allow-
84
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
TABLE 1 Hydrolysis of Lysozyme by 5-fold Distilled
6 N Hydrochloric
Residues Expt.
No.
Lysoeyme
sample, mg
Hydrolysis temperature,
4.8 4.9 4.63 3.48 3.92 Residues actually
found
on basis
Acid
of 6 lysine
Tryptophan
“C
90-92 85-87 88-89 88-89 88-89 in lysozyme :
r&dues
Histidine
3.5 2.13 2.64 3.5 5.6 6
1.05 0.52 1.16 Not determined Not determined 1
ing the solution to stand not less than 24 hr before taking the spectral characteristics. It seems to be the same with the products of lysozyme photoxidation. Since tyrosine residues are not affected under the chosen photoxidation conditions (13)) the ratio tyrosine: tryptophan may change only because of a possible destruction of tryptophan residues. Hence in this case R can have only the following values: 3:6 = 0.5 3~5 = 0.6 3:4 = 0.75, etc. It is seen from Table 2 that R did not rise above 0.5 for any lysozyme photoxidation product. Thus we may conclude that tryptophan in X, Y, and Z was fully retained. The pronase hydrolysis, however, and differential UV spectra showed that this was not the case. It became evident that in tryptophan photoxidation the modification made it impossible to detect the difference between ordinary and photomodified tryptophan residues hy the Bencze and Schmid technique. The results obtained were TABLE 2 Analysis of Lysozyme and Products of Its Photoxidation bv Modified Spectrophotometric Method Protein
Native lysozyme
Solution standing time before taking spectral characteristics, hr
Found
8 CdO.
Found
R CdC.
Immediately 24
-14.1 -9.95
-9
0.2 0.5
0.5
X
3 24
-11.65 -9.25
-9
0.44 0.5
0.5
Y
3 24
-11.8 -9.9
-9
0.34 0.5
0.5
Z
3 24
-12.47 -8.87
-9
0.3 0.5
0.5
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
85
rather close to those for native lysozyme. This method is limited in its application even for proteins with nonmodified tryptophan and tyrosine since its accuracy depends on inambiguity of protein spectral characteristics in the solution, which in turn are greatly influenced by many factors not always possible to be taken into consideration (14-17). Hydrolysis of lysozyme and of the products of its photoxidation by pronase has attracted our attention. Ferrini et al. (2) point out that in a week’s incubation pronase splits up about 90% of lysozyme peptide bonds in addition to displaying no specificity. Protein samples with pronase were incubated for 14 days. As is evident from the data reported below (Table 4) the tryptophan under study was released in full. (Further experiments have shown that under these conditions pronase activity drops sharply, almost twice, during the first 100 hr of incubation with a very slight subsequent change). Hydrolysis by pronase has been studied on native lysozyme. After incubation in 0.2 M (CH&OO),Ca the mixture was dried, then dissolved in 1 ml of a suitable buffer and analyzed on a 15 cm column of the amino acid analyzer. The analytic curves were, however, very distorted and not good for calculation. The analysis of the test mixture of the basic amino acids with an addition of the proper amount of (CH,COO),Ca resulted in the same distortions. In fact, the excess of Ca+? obstructed the analysis, as could have been expect,ed, because many amino acids form complexes with Cat2 (18). The effect of Cac2, however, has been found to be stepwise. Up to a Ca+2 content of 0.32 m.M the curves were conventional, but then distortions appeared spontaneously. It is interesting to note that the configuration of this distorted curve for amino acid analysis of native lysozyme was similar to the one obtained for UV-irradiated lysozyme by Ferrini et al. (2). In either case an extra peak appeared next to that of lysine, and was incompletely separated from the latter. Although the amounts of Ca+Z present in the hydrolyzates of Ferrini et al. did not produce any deviation under the conditions, the Ca+2 effect of these authors cannot be neglected, since various samples of ion exchangers used for amino acid aanlysis may react differently to the presence of Caf2. Therefore it became necessary to liberate the hydrolyzate from Cat”. After unsuccessful attempts to bind Ca+2 in carbonate or by EDTA, an Amberlite CG-120 resin was used. Applying ammonia of various concentrations to elute the amino acid mixture from the resin, we obtained a hydrolyzate that gave a perfect curve in amino acid analysis. Table 3 shows that quantitative and proportional elution is attained only by applying 9 N NH,OH (10 ml/l ml of resin). Under elution by less concentrated NH,OH, amino acids are unproportionally lost, sometimes
86
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
TABLE 3 Desalting of Synthetic Mixture of Basic Amino Acida on a 1 X 3 cm Amberlite CG-120 Column Amino elution
Coi?t
Amino
acid
mixture before “d,y,
Tryptephan Lysine Histidine Arginine
1 1.04 0.99 1.04
acids in mixture after by ammonia of various
4N,ZOml rmole
0.74 0.67 0.65 0.65
4 N, 50 ml
yield,
74 64.5 65.5 62.5
‘%
rmole
1.11 0.95 0.85 0.92
yield,
desalting under concentrations 8N,30ml
y0
111 92 86 88.5
rmole
QN,30ml
yield,
1.17 0.98 0.93 1.04
%
117 95 94 100
rmole
yield,
1.03 1.1 1.03 1.06
70
103 106 104 102
up to 37%, tryptophan being lost less than others. Pronase autolyzate control showed no peaks in the range of basic amino acids. These facts being taken in consideration, analyses of native lysozyme and of the products of its photoxidation were carried out. The results are given in Table 4. There being 6 tryptophan residues in lysozyme, the actual yield for tryptophan after pronase hydrolysis is 96%. Therefore corresponding corrections have been made for the modification products as well. Hence the number of tryptophan residues for Z is 4.76, Y 5.95, and X 5.87, or approximately 5, 6, and 6, respectively. Thus only pronase hydrolysis revealed the difference in tryptophan content in native lysozyme and in one of the lysozyme photoxidation products. Thus the tryptophan residue destroying in Z was revealed by amino acid analysis after pronase hydrolysis and was confirmed by preliminary data of the differential UV spectrum and the peptide map. TABLE 4 Amino Acid Analysis of Pronase Hydrolyzates of Lysozyme and Its Photexidation Products Residues Protein
Lysozyme Average Z Average Y Average X
Tryptophan
5.7 5.64 5.9 5.75 4.5 4.63 4.56 6 5.4 5.7 5.63
found
cm basis Hitidiie
0.88 0.7 0.79 0.77 0.45 0.61 0.58 0.33 0.45 0.38
of 6 lyaine
residues Arghhe
10.2 11.4 9.3 10.3 11.5 7.6 9.6 12 10.7 11.3 11.4
TRYPTOPHAN
DETERMINATION
IN
87
LYSOZYME
TABLE 5 Yields of Amino Acids after Desalting from Ca+’ on an Amberlite CG-120 Column (1 X 3 cm) (elution by 30 ml 9 N NHnOH) Amino
acid
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Yield,
70
10.9 102 101 95 98 100 97 97 121 (?) 84 97.5 10’2 100 100
The procedure for desalting basic amino acids with elution by 9 N NH,OH can also be applied to other amino acids, as is illustrated in Table 5. The low yield of aspartic acid is apparently accounted for by the slight sorbtion during the passage through the column with salt mixture. Methionine is partly destroyed on resin, the desalting being carried out without thiodiglycol. The causes of too high valine yield having occurred twice have not been investigated by us. A typical curve of amino acid analysis of pronase hydrolyzates is shown in Figure 3. It is of the same configuration for both native lysozyme and its photoxidation products. The curve has two extra peaks-l and 2-in contrast to the standard one. They are evidently due to the fact that the histidine and arginine contents in all the preparations studied (cf. Table 4) are too low as compared with the theoretical values (1 and 11 residues, respectively). Since, according to the preliminary data (peptide map), histidine is not affected during photoxidation, and both extra peaks are present on every curve including that of lysozyme, it may be assumed that peak 1 corresponds to histidine-containing underhydrolyzed peptides, whereas peak 2 corresponds to arginine-containing ones. Moreover, peak 2 has a sufficiently stable relative value in all pronase hydrolyzates, whiIe peak 1 increases relative to the other amino acid peaks in the row, namely, lysozyme - 2 - Y - X. The values established for histidine decreases with the same regularity (cf. Table 4)) confirming the reason for the appearance of peak 1. The sum total of histi-
88
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
Fm. 3. Typical curve of amino acid analysis of pronase hydrolyeates of lysosyme and ita photoxidation products: (1 and 2) extra peaks, compared with standard curve. Broken line indicates distorted curve obtained in presence of large quantity of Ca+“.
dine and of the substance represented by peak 1 (histidine being used for peak 1 calculation) is close to one residue.
color yield
SUMMARY
A comparative study was made of three techniques of tryptophan determination in lysozyme and its photoxidation products: mild acid hydrolysis (85-90°, 15 hr), pronase hydrolysis, and direct spectrophotometric determination. Satisfactory data have been obtained only after pronase hydrolysis. The technique for hydrolyzate treatment and its analysis have been worked out. This technique was used for tryptophan determination in three lysozyme photoxidation products. The mild acid hydrolysis has been found unsuitable owing to incomplete hydrolysis. The direct spectrophotometric determination has been modified as applied to lysozyme, and satisfactory results have been obtained. But this technique is unsuitable for tryptophan determination in photoxidized lysozymes as it does not permit differentiation of native from photoxidized tryptophan residues in protein. REFERENCES 1. MONIER, R., AND JUTISZ, M., Bull. Sot. Chim. Biol. 32, 228 (1950). 2 FERRINI, U., ZITO, R., AND CAPUTO, A,. in “Atti de1 III symposium internationale sul lisoeima di Fleming,” I Sezione (b) Biologia de1 lisosima, Milan, 1964, p. 36.
TRYPTOPHAN
DETERMINATION
IN
3. BENCZE,
6.
7. 8. Y.
10. 11. 12. 13. 14. 15. 16. 17.
89
W. L., AND SCHMID, K., And. Chem. 29, 1193 (1957). N. A., STEPANOVA, N. B., LAPUK, V. KH., AND C~RKASOV, I. A., Molekularnaya biol. (USSR) 1, 47 (1967). GURNANI, S., ARIF~DDIN, M., AND AUGUSTI, K., Photochem. Photobiol. 5, 495 (1966). FERRINI, U., Arch. Biochem. Biophys. 107, 126 (1964). ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164, 1 (1946). KRAVCHENKO, N. A., KLEOPINA, G. V., AND KAVERZNEVA, E. D., Biokhimiya 30, 534 (1965). KRAVCHENKO, N. A., KLEOPINA, G. V., AND KAVERZNEVA, E. D., Biokhimiya 30, 713 (1965). KAVERZNEVA, E. D., AND RASSULIN, J. A., Prikl. Biokhim. Mikrobiol. 2, 51 (1966). NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M., J. Biochem. 48, 453 (1960). OPIENSKA-BLAUTH, J. CHAREZINSKI, M., SANECKA, M., AND BR~TJSZKIEWCZ, H., J. Chromatog. 7, 321 (1962). WEIL, L., Arch. Biochem. Biophys. 110, 57 (1965). HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 237, 3418 (1962). WILLIAMS, E. J., HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3574 (1965). WILLIAMS, E. J., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3580 (1965). TOJO, T., HAMAGUCHI, K., IMANISHI, M., AND AMANO, T., J. Biochem. 60, 538
4. KRAVCHENKO, 5.
LYSOZYME
(1966)
18. HARDEL,
M.,
Z. Physiol.
Chem.
346,
224
(1966).
24, 80-89 (1968)
BIOCHEMISTRY
On the Tryptophan
Determination Photoxidation
V. KH. LAPUK, The
in Lysozyme
and
Its
Products
L. A. CHISTYAKOVA,
N. A. KRAVCHENKO
AND
Laboratory of Protein Chemi&ry, Institute USSR Academy of Sciences, Leninski prospekt
of
Organic
47, Moscow
Chemistry,
B-334, USSR
Received July 28, 1967 All the existing techniques of tryptophan determination in proteins may be classified into two large groups: (1) determination of tryptophan after its isolation from protein by means of chemical or enzymic hydrolysis and (2) direct determination without preliminary isolation. We applied to lysozyme photoxidation products techniques described in the literature-mild acid hydrolysis (1)) hydrolysis by pronase (2), and direct spectrophotometric method (3). These techniques were tested on native lysozyme as well as on the previously isolated products of its photoxidation by visible light (4). The tests were particularly important in this case as photoxidation was carried out to decompose tryptophan. Tryptophan photoxidation is known to be very complicated, with a gradual indole ring degradation (5). The effect of the modification of the major lysozyme chromophores on lysozyme UV spectrum, as well as the stability of the photoxidized tryptophan residues during analysis, have remained obscure. According to Ferrini (6), the products of histidine photoxidation in lysozyme change back into histidine during the usual acid hydrolysis. This fact led to a very cautious estimation of the results of the analysis of tryptophan photoxidation products. The three techniques mentioned above were chosen as the simplest, since tryptophan is not destroyed. METHODS Lysozyme was obtained by direct crystallization according to Alderton and Fevold (7), with subsequent desalting on Sephadex G-25, and by lyophilization from acetic acid (8). The products of lysozyme photoxidation were obtained by the technique described above (4) ; they are denoted by symbols X, Y, and Z, the residual activity on Micrococcus lysode-ikticus (9) being 73, 33, and 12%, respectively. Pronase was prepared in our laboratory, its activity being 0.116 X 10 1 proteinase units per milligram (10). 80
Hydrolysis
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
of lysozyme
by repeatedly
distilled
hydrochloric
81
acid (1).
5 mg of lysozyme was dissolved in 5 ml of 5-fold distilled HCI (5.7 N) . The solution was then poured into a tube, the air evacuated, and hydrolysis allowed to proceed for 15 hr at 85-90”. After drying in vacua over NaOH, the substance was analyzed by an amino acid analyzer (Czechoslovakia) . Hydrolysis
of lysoxyme
and the products
of its photoxidation
by pro-
nase (2). 44.5
mg of protein was dissolved in 3.5-4 ml of 0.2 M solution of (CH,COO)&a (ll), at pH set to 28.3, and 0.1 ml of 2.5 mg pronase solution in 1 ml of the same solvent was added. This mixture was then frozen in a Pyrex tube at -7O*, the air evacuated, and the mixture defrosted, refrozen, re-evacuated, and incubated for 2 weeks at 37-38“. The hydrolyzate was filtered and placed on a column with Amberlite CG-120, H+-form (1 X 3 cm) ; the column was washed with water until a neutral reaction was obtained and eluted by 30 ml 9N NH,OH. The eluate was dried in vacua over H,SO,. The hydrolyzate was analyzed by the amino acid analyzer. Since all the three products of photoxidation of lysozyme dissolved incompletely, the calculation of amino acid analyses on a sample was rather difficult. Therefore the calculation was made on 6 lysine residues for basic amino acids (tryptophan, lysine, histidine, arginine) and on 3 tyrosine residues (the real amount of these residues in lysozyme) for the remaining amino acids. Spectrophotometric determination of tryptophan in lysozyme and .in the products of lysozyme photoxidation by a modified technique of Bencze and Schmid (3). A protein solution of specific concentration
(within the range of 0.25-0.3 mg/ml) was prepared in 0.1 N NaOH and allowed to stand for 24 hr at 20’. After that, OD,,, and OD,,, were determined. The two points obtained were connected by a straight line in the coordinate system given in Figure 1. Here the optimum scale for calculation was as follows: ordinate, 25 mm = 0.1 OD, abscissa, 5 mm = 1 mp. Then the value s = (AOD/Ax) X lo3 Ohm was calculated. In accordance with the data in the literature (3) the dependence plot of S on R was drawn (Fig. 2)) where R is the molar tyrosine: tryptophan ratio. From Figure 2 and the calculated value S, the value R for a given protein sample, i.e., the tyrosine:tryptophan ratio, was found, either as an integer or a fractional number. Considering that tyrosine was not
82
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENEO
modified in our case, the number of tryptophan residues was determined assuming the number of tyrosine residues to be 3. RESULTS AND DISCUSSION
The routine acid hydrolysis of protein (6 N HCl, 105-llO’,’ 24 hr) is known to be unsuitable for the determination of tryptophan, the latter being fully destroyed in the course of hydrolysis (12). However in lysozyme hydrolysis with air thoroughly removed and HCl distilled in glass, the tryptophan peak appeared in many cases, its amount being not more than 3 residues. It is of interest that in these cases an unidentified peak was always present before the tryptophan peak, the ratio of their
FIG. 1. Tryptophan determination by spectrophotometric scale: 0.1 OD = 25 mm, 1 ma = 5 mm.
technique. Optimum
quantities being approximately 1:3. This suggested that the extra peak corresponded to some product of tryptophan destruction, slightly more acidic than tryptophan itself, and that it was possible to find conditions of acid hydrolysis under which tryptophan was not destroyed. This was confirmed by the data of Monier and Jutisz (1). They hydrolyzed lysozyme by repeatedly distilled hydrochloric acid at 85-90” without destroying tryptophan. We used the Monier and Jutisz technique and the results are given in Table 1. Table 1 shows that the figures on tryptophan are too low and diversified. Moreover, the extra peak presumably corresponding to the incompletely hydrolyzed peptides appears on the curves of amino acid analysis of the hydrolyzates between the lysine peak and that of histidine. All these facts indicate that the hydrolysis is not complete. The hydrolyzates obtained
are of dark,
almost
black,
color and have a gumlike
consist-
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
83
I5
i0
5
FIO. 2. Tyrosine:tryptophan relationship determination on plot drawn in accordance with data of Bencse and Schmid (3).
ency. The technique is therefore not suitable for tryptophan determination. The direct spectrophotometric method of Bencze and Schmid (3) for tryptophan (and tyrosine) determination was attempted and the results obtained for native lysozyme and the products of its photoxidation are given in Table 2. It follows from Table 2 that values of S and R close to the calculated ones for native lysozyme can be obtained only after allow-
84
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
TABLE 1 Hydrolysis of Lysozyme by 5-fold Distilled
6 N Hydrochloric
Residues Expt.
No.
Lysoeyme
sample, mg
Hydrolysis temperature,
4.8 4.9 4.63 3.48 3.92 Residues actually
found
on basis
Acid
of 6 lysine
Tryptophan
“C
90-92 85-87 88-89 88-89 88-89 in lysozyme :
r&dues
Histidine
3.5 2.13 2.64 3.5 5.6 6
1.05 0.52 1.16 Not determined Not determined 1
ing the solution to stand not less than 24 hr before taking the spectral characteristics. It seems to be the same with the products of lysozyme photoxidation. Since tyrosine residues are not affected under the chosen photoxidation conditions (13)) the ratio tyrosine: tryptophan may change only because of a possible destruction of tryptophan residues. Hence in this case R can have only the following values: 3:6 = 0.5 3~5 = 0.6 3:4 = 0.75, etc. It is seen from Table 2 that R did not rise above 0.5 for any lysozyme photoxidation product. Thus we may conclude that tryptophan in X, Y, and Z was fully retained. The pronase hydrolysis, however, and differential UV spectra showed that this was not the case. It became evident that in tryptophan photoxidation the modification made it impossible to detect the difference between ordinary and photomodified tryptophan residues hy the Bencze and Schmid technique. The results obtained were TABLE 2 Analysis of Lysozyme and Products of Its Photoxidation bv Modified Spectrophotometric Method Protein
Native lysozyme
Solution standing time before taking spectral characteristics, hr
Found
8 CdO.
Found
R CdC.
Immediately 24
-14.1 -9.95
-9
0.2 0.5
0.5
X
3 24
-11.65 -9.25
-9
0.44 0.5
0.5
Y
3 24
-11.8 -9.9
-9
0.34 0.5
0.5
Z
3 24
-12.47 -8.87
-9
0.3 0.5
0.5
TRYPTOPHAN
DETERMINATION
IN
LYSOZYME
85
rather close to those for native lysozyme. This method is limited in its application even for proteins with nonmodified tryptophan and tyrosine since its accuracy depends on inambiguity of protein spectral characteristics in the solution, which in turn are greatly influenced by many factors not always possible to be taken into consideration (14-17). Hydrolysis of lysozyme and of the products of its photoxidation by pronase has attracted our attention. Ferrini et al. (2) point out that in a week’s incubation pronase splits up about 90% of lysozyme peptide bonds in addition to displaying no specificity. Protein samples with pronase were incubated for 14 days. As is evident from the data reported below (Table 4) the tryptophan under study was released in full. (Further experiments have shown that under these conditions pronase activity drops sharply, almost twice, during the first 100 hr of incubation with a very slight subsequent change). Hydrolysis by pronase has been studied on native lysozyme. After incubation in 0.2 M (CH&OO),Ca the mixture was dried, then dissolved in 1 ml of a suitable buffer and analyzed on a 15 cm column of the amino acid analyzer. The analytic curves were, however, very distorted and not good for calculation. The analysis of the test mixture of the basic amino acids with an addition of the proper amount of (CH,COO),Ca resulted in the same distortions. In fact, the excess of Ca+? obstructed the analysis, as could have been expect,ed, because many amino acids form complexes with Cat2 (18). The effect of Cac2, however, has been found to be stepwise. Up to a Ca+2 content of 0.32 m.M the curves were conventional, but then distortions appeared spontaneously. It is interesting to note that the configuration of this distorted curve for amino acid analysis of native lysozyme was similar to the one obtained for UV-irradiated lysozyme by Ferrini et al. (2). In either case an extra peak appeared next to that of lysine, and was incompletely separated from the latter. Although the amounts of Ca+Z present in the hydrolyzates of Ferrini et al. did not produce any deviation under the conditions, the Ca+2 effect of these authors cannot be neglected, since various samples of ion exchangers used for amino acid aanlysis may react differently to the presence of Caf2. Therefore it became necessary to liberate the hydrolyzate from Cat”. After unsuccessful attempts to bind Ca+2 in carbonate or by EDTA, an Amberlite CG-120 resin was used. Applying ammonia of various concentrations to elute the amino acid mixture from the resin, we obtained a hydrolyzate that gave a perfect curve in amino acid analysis. Table 3 shows that quantitative and proportional elution is attained only by applying 9 N NH,OH (10 ml/l ml of resin). Under elution by less concentrated NH,OH, amino acids are unproportionally lost, sometimes
86
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
TABLE 3 Desalting of Synthetic Mixture of Basic Amino Acida on a 1 X 3 cm Amberlite CG-120 Column Amino elution
Coi?t
Amino
acid
mixture before “d,y,
Tryptephan Lysine Histidine Arginine
1 1.04 0.99 1.04
acids in mixture after by ammonia of various
4N,ZOml rmole
0.74 0.67 0.65 0.65
4 N, 50 ml
yield,
74 64.5 65.5 62.5
‘%
rmole
1.11 0.95 0.85 0.92
yield,
desalting under concentrations 8N,30ml
y0
111 92 86 88.5
rmole
QN,30ml
yield,
1.17 0.98 0.93 1.04
%
117 95 94 100
rmole
yield,
1.03 1.1 1.03 1.06
70
103 106 104 102
up to 37%, tryptophan being lost less than others. Pronase autolyzate control showed no peaks in the range of basic amino acids. These facts being taken in consideration, analyses of native lysozyme and of the products of its photoxidation were carried out. The results are given in Table 4. There being 6 tryptophan residues in lysozyme, the actual yield for tryptophan after pronase hydrolysis is 96%. Therefore corresponding corrections have been made for the modification products as well. Hence the number of tryptophan residues for Z is 4.76, Y 5.95, and X 5.87, or approximately 5, 6, and 6, respectively. Thus only pronase hydrolysis revealed the difference in tryptophan content in native lysozyme and in one of the lysozyme photoxidation products. Thus the tryptophan residue destroying in Z was revealed by amino acid analysis after pronase hydrolysis and was confirmed by preliminary data of the differential UV spectrum and the peptide map. TABLE 4 Amino Acid Analysis of Pronase Hydrolyzates of Lysozyme and Its Photexidation Products Residues Protein
Lysozyme Average Z Average Y Average X
Tryptophan
5.7 5.64 5.9 5.75 4.5 4.63 4.56 6 5.4 5.7 5.63
found
cm basis Hitidiie
0.88 0.7 0.79 0.77 0.45 0.61 0.58 0.33 0.45 0.38
of 6 lyaine
residues Arghhe
10.2 11.4 9.3 10.3 11.5 7.6 9.6 12 10.7 11.3 11.4
TRYPTOPHAN
DETERMINATION
IN
87
LYSOZYME
TABLE 5 Yields of Amino Acids after Desalting from Ca+’ on an Amberlite CG-120 Column (1 X 3 cm) (elution by 30 ml 9 N NHnOH) Amino
acid
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Yield,
70
10.9 102 101 95 98 100 97 97 121 (?) 84 97.5 10’2 100 100
The procedure for desalting basic amino acids with elution by 9 N NH,OH can also be applied to other amino acids, as is illustrated in Table 5. The low yield of aspartic acid is apparently accounted for by the slight sorbtion during the passage through the column with salt mixture. Methionine is partly destroyed on resin, the desalting being carried out without thiodiglycol. The causes of too high valine yield having occurred twice have not been investigated by us. A typical curve of amino acid analysis of pronase hydrolyzates is shown in Figure 3. It is of the same configuration for both native lysozyme and its photoxidation products. The curve has two extra peaks-l and 2-in contrast to the standard one. They are evidently due to the fact that the histidine and arginine contents in all the preparations studied (cf. Table 4) are too low as compared with the theoretical values (1 and 11 residues, respectively). Since, according to the preliminary data (peptide map), histidine is not affected during photoxidation, and both extra peaks are present on every curve including that of lysozyme, it may be assumed that peak 1 corresponds to histidine-containing underhydrolyzed peptides, whereas peak 2 corresponds to arginine-containing ones. Moreover, peak 2 has a sufficiently stable relative value in all pronase hydrolyzates, whiIe peak 1 increases relative to the other amino acid peaks in the row, namely, lysozyme - 2 - Y - X. The values established for histidine decreases with the same regularity (cf. Table 4)) confirming the reason for the appearance of peak 1. The sum total of histi-
88
LAPUK,
CHISTYAKOVA,
AND
KRAVCHENKO
Fm. 3. Typical curve of amino acid analysis of pronase hydrolyeates of lysosyme and ita photoxidation products: (1 and 2) extra peaks, compared with standard curve. Broken line indicates distorted curve obtained in presence of large quantity of Ca+“.
dine and of the substance represented by peak 1 (histidine being used for peak 1 calculation) is close to one residue.
color yield
SUMMARY
A comparative study was made of three techniques of tryptophan determination in lysozyme and its photoxidation products: mild acid hydrolysis (85-90°, 15 hr), pronase hydrolysis, and direct spectrophotometric determination. Satisfactory data have been obtained only after pronase hydrolysis. The technique for hydrolyzate treatment and its analysis have been worked out. This technique was used for tryptophan determination in three lysozyme photoxidation products. The mild acid hydrolysis has been found unsuitable owing to incomplete hydrolysis. The direct spectrophotometric determination has been modified as applied to lysozyme, and satisfactory results have been obtained. But this technique is unsuitable for tryptophan determination in photoxidized lysozymes as it does not permit differentiation of native from photoxidized tryptophan residues in protein. REFERENCES 1. MONIER, R., AND JUTISZ, M., Bull. Sot. Chim. Biol. 32, 228 (1950). 2 FERRINI, U., ZITO, R., AND CAPUTO, A,. in “Atti de1 III symposium internationale sul lisoeima di Fleming,” I Sezione (b) Biologia de1 lisosima, Milan, 1964, p. 36.
TRYPTOPHAN
DETERMINATION
IN
3. BENCZE,
6.
7. 8. Y.
10. 11. 12. 13. 14. 15. 16. 17.
89
W. L., AND SCHMID, K., And. Chem. 29, 1193 (1957). N. A., STEPANOVA, N. B., LAPUK, V. KH., AND C~RKASOV, I. A., Molekularnaya biol. (USSR) 1, 47 (1967). GURNANI, S., ARIF~DDIN, M., AND AUGUSTI, K., Photochem. Photobiol. 5, 495 (1966). FERRINI, U., Arch. Biochem. Biophys. 107, 126 (1964). ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164, 1 (1946). KRAVCHENKO, N. A., KLEOPINA, G. V., AND KAVERZNEVA, E. D., Biokhimiya 30, 534 (1965). KRAVCHENKO, N. A., KLEOPINA, G. V., AND KAVERZNEVA, E. D., Biokhimiya 30, 713 (1965). KAVERZNEVA, E. D., AND RASSULIN, J. A., Prikl. Biokhim. Mikrobiol. 2, 51 (1966). NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M., J. Biochem. 48, 453 (1960). OPIENSKA-BLAUTH, J. CHAREZINSKI, M., SANECKA, M., AND BR~TJSZKIEWCZ, H., J. Chromatog. 7, 321 (1962). WEIL, L., Arch. Biochem. Biophys. 110, 57 (1965). HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 237, 3418 (1962). WILLIAMS, E. J., HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3574 (1965). WILLIAMS, E. J., AND LASKOWSKI, M., JR., J. Biol. Chem. 240, 3580 (1965). TOJO, T., HAMAGUCHI, K., IMANISHI, M., AND AMANO, T., J. Biochem. 60, 538
4. KRAVCHENKO, 5.
LYSOZYME
(1966)
18. HARDEL,
M.,
Z. Physiol.
Chem.
346,
224
(1966).