J. Mol. Biol. (1986) 190; 647-649
Unfolding
Rates of Globular Proteins Determined by Kinetics of Proteolysis
A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50°C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 5O”C, and those of lysozyme, RNase A and P-lactoglobulin at pH 8 and 4O”C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.
The idea that the digestion of a small globular protein with a protease proceeds via the unfolded state (D) rather than the folded state (N) of the protein has become accepted gradually (Linderstrom-Lang et al., 1938; Matthyssens et al., 1972; Imoto et al., 1974, 1976; Pace & Barret, 1984). Most of the published work has been based on the assumption that the N+D transition of a protein (eqn (1)) is much faster than the rate of subsequent digestion with a protease (E):
J&J?&
seemed to be saturated at high concentrations of thermolysin. Similar saturation behaviors have been reported without recognition in the proteolysis of lysozyme with pepsin (Matthyssens et al.; 1972) and with Nagarse (subtilisin BPN’: Imoto et al., 1976). Assuming steady state (d[D]/dt = 0 and d[ED]/dt = 0), the over-all rate of digestio:n in equation (1) is expressed as:
’ = ks’ED1 ED 4 E + peptide.
= (k,+k,)/k,
+ (k,/k,)[E]
(1)
(f&,
Here, we show that the NG D transition is not always faster than the rate of the subsequent the unfolding rate digestion and, as a result, constant (k, in eqn (1)) for some globular proteins can be determined by a digestion method under physiological conditions. egg-white lysozyme (0.25 mg/ml) was Hen digested with various concentrations (0.29 to 2.87 PM) of thermolysin in 0.1 M-NH,HCO, containing 2 mmCaCl, at pH 8.0 and 50°C. Samples (50 ~1) were taken at appropriate intervals and analyzed by use of carboxylic cation-exchange high-pressure liquid chromatography. The column (TSKgel CM3SW, 4 mm x 300 mm) was eluted with 0.05 Msodium phosphate buffer containing 0.25 M-NaCl at pH 7.0 and a flow-rate of 0.8 ml per minute, and the of the effluent at 210nm was absorbance monitored. During the reaction, only two peaks (one excluded and one non-digested lysozyme peak) appeared in the chromatograph and no intermediate peak was observed, i.e. “all or none” digestion took place, as reported (Imoto et al., 1974, 1976). The decrease of lysozyme, thus monitored, followed pseudo first-order kinetics, at least until the second half-life period. The apparent first-order increased non-linearly with rate constant, i&r, increase of the concentration of thermolysin, and
where K, (= lc,/rE,) is the equilibrium constant for the Ne D transition of the protein and K, (= (k,+ IC,)/ks) is the Michaelis constant for the proteolysis. Since [D] and [ED] are considered to be negligible under physiological conditions, [N] and [E] in equation (2) are essentially equal to the concentrations of non-digested protein and total protease ([E-r]), respectively. Therefore, iE,rr and wapp are expressed as:
(3)
Equation (4) suggests that double reciprocal -plots give a straight of bpp versus protease concentration line, with l/k, as the intercept of l/lc,,, with lj[Er] being zero if the digestion of the protein proceeds only via the unfolded state. As shown in Figure l(a), the digestion of lysozyme with thermolysin at pH 8.0 and 50°C was such a case, and the unfolding rate constant (k, ) of lysozyme was estimated to be
647 002%2836/86/16064703
$03.00/O
0
1986 Academic
Press Inc.
(London)
Ltd.
648
T. Imoto et a!.
9.2( +@4) x low4 s-l. In order to confirm that the i%l value obtained here is the unfolding rate constant of lysozyme, the following experiments carried were out. (1) When reduced and S-S(trimethylated amino) propylated lysozyme (Okazaki et al., 1985), which is completely unfolded and therefore lacks the N+ D transition, was digested with thermolysin under the same conditions as used for native lysozyme, IC& was proportional to the concentration of thermolysin and no saturat,ion behavior of Jcapp was observed. (2) If we are looking at Ic, of lysozyme, the value should not be affected by changing the protease. The digestion of lysozyme with Pronase in 0.1 MNH,HCO, at pH 8.0 and 50°C showed the saturation behavior of Knapprelated to the concentration of Pronase (Fig. l(a)), and k, was estimated to be 9.1 (+0-B) x lop4 s-l (Table 1). (3) As shown in Figure l(b), the unfolding rate constants of lysozyme at pH 8.0 and 50°C in the presence of various concentrations of guanidinium chloride were determined spectrophotometrically by the decrease in absorbance of lysozyme at 301 nm on rapid changing of the concentration of Gu . HCI from zero, and were extrapolated t’o zero concentration of denaturant (Tanford et al., 1966) to give a lE, value of 9.1 (i3.7) x 10e4 s-l. Although this linear extrapolation may not give a precise value for Ic, at. zero concentration of Gu . HCl, as discussed by Pace & Vandenburg (1979), the value was consistent with those obtained from the digestion method. All of these results clearly indicat’e that the digestion of lysozyme with thermolysin as well as with Pronase proceeds only via t’he unfolded state (D) of lysozyme and, furthermore, the unfolding rate constant (k,) of lysozyme can be determined by the digestion method. In order to examine the applicability of the digestion method the determination of the unfolding rate constant of other proteins, bovine pancreatic ribonuclease A (RNase A) was digested with thermolysin and with Pronase at pH 8.0 and 50°C. The rates of digestion of RNase A were determined as described for lysozyme, except that the column of TSKgel CM-3SW was eluted with O.O5M-sodium phosphate buffer (pH 7) containing 0.03 w-NaCl. Apparent first-order rate constants (k,,,) for digest,ions of RNase A were again saturated with the concentrations of thermolysin and Pronase, and the k, values obtained with each protease were again identical; as shown in Table 1. Furthermore, the unfolding rate constant for RNase A obtained by the relaxation method (Fig. l(b) and Table 1) was identical to those obtained by the digestion method. Therefore, the digestion method for the determination of the unfolding rate constant of globular proteins is considered to be general. The unfolding rate constants of lysozyme, RNase A and P-lactoglobulin (/?-lactoglobulin B designated by Piez et al., 1961) at pH 8.0 and 40°C determined by the digestion method using various proteases are shown in Table 1. The digestion of p-
lactoglobulin was followed by anion-exchange higbpressure liquid chromat’ography on a column of TSKgel DEAE-5PW (4.6 mm x 150 mm), which was eluted with 0.05 M-sodium phosphate buffer containing 0.12 M-KaCl at pH 9.0 and a flow-r&e of 0.8 ml per minute. In the case of lysozrme, achymotrypsin, Pronase and thermolysin gave, within experimental error; identical unfolding rate constant8 (k, = 2.2 x 10S4 to 2.3 x lop4 5-l). However, the determination of the Ic, value for lysozyme by digestion with trypsin was not possible, because lysozyme is hardly digested with t#rypsin at plf 8.0 and 40°C (Imoto et al., 1974). This result suggest,s that Ic, and/or E, in equation (1) are very small in this case. In the ease of P-lactoglobuiin, essentially identical lc, values (7.2 x lo-” t’o 8.0 x 10e4 s-l) were obtained using three different proteases. As for RNase A, only digestion with Pronase showed the saturation kinetics, and the 1%1value of RNase A
1 20000
2 P 0 :l0000
0
I
I
I
I
I
20
40
60
80
100
I/CProteosel
(mllmg)
(0)
I
1
I
ot -I I-
I
I I
I 2 Gu HCI concn it.4I
I 3
(b)
Figure 1. (a) Double reciprocal plots of k,,, vwsu~s the concentration of protease for the digestion of lpsozvme with thermolysin (0) and with Pronase (A) at pN X.0 and 50°C. (b) Linear extrapolations of log k1 for the KJ= D transitions of lysozyme (0) and RR-ase A ( pH 8,O and 50°C from those at various concentrations of Gu HCI to those at zero concentration of denaturant.
649
Letters to the Editor
Table 1 The unfolding
Protein
Temperature (“C)
rate constants
9.1+ 3.7
RNase $
50 40
s.9+2,9
jj-Lactoglobulin
40
j’ In 0.1 M-TIiS HCl buffer. $ In 0.1 M-NH,HCO,. $ In the presence of 2 mM-C&l,. 11The rate constant could not be determined
proteins Digestion
Relaxation method? (x 10-b s-1)
50 40
Lysozyme
(k,) of globular
at pH 8.0 method ( x 10e4 s- ‘)$
Pronase
cl-Chymotrypsin
9.2kO.4 2.2&04
9.150.6 2.3f0.4
2.2kO.2
10.1+0,4 II 7.2kO.6
9.8+0.6 4.4kO.4
II
7.9kO.4
&O&O-4
Thermolysin§
Trypsin
II
because of little or no digestion
was found to be 4.4 x 10m4 s-l at pH 8.0 and 40°C. Digestion of RNase A with a-chymotrypsin did not proceed detectably, and that with thermolysin was too slow to obtain any reliable measure of k, for RNase A at this temperature. These observations are in marked contrast to those in the cases of lysozyme and B-lactoglobulin, and to that in the case of the thermolysin digestion of RNase A at 50°C. Evidently, the susceptibility of unfolded substrate and the activity of the protease at certain temperatures are important factors in determination of the unfolding rate constant of proteins by the digestion method. Of course, in some cases the folded proteins are digested with certain proteases to a limited extent. In such cases equation (4) no longer holds and the unfolding rate constant of the protein cannot be determined by digestion with such proteases. Therefore, the appropriate protease must be chosen for digestion of the protein in question. It may be considered good evidence of a reliable determination of Xe, if at least two proteases give saturation kinetics and consistent k, values. Since there have been no reliable methods for the determination of the unfolding rates of globular proteins under physiological conditions, where the concentrations of unfolded proteins are extremely small, the present digestion method should give a critical insight into the elucidation of protein stability. Moreover, it was shown that digestion with proteases proceeds via mainly the unfolded states of proteins. The finding that the stability of proteins against proteases is related to unfolding rates as well as to the denaturation equilibria of proteins may aid the molecular design of proteins Edited
for effective use as drugs, industrial catalysts, so on, by means of chemical modifications genetic engineering.
and and
Taiji Imoto Hidenori Yamada Tadashi Ueda Faculty of Pharmaceutical Sciences Kyushu University 62 Maidashi, Higashi-ku, Fukuoka 812, Japan Received 14 April 1986
References Imoto, T., Fukuda, K. & Yagishita, K. (1974). Biochim. Biophys. Acta, 336, 264-269. Imoto, T., Fukuda., K. & Yagishita, K. (1976). J. Biochem. (Tokyo), 80, 1313-1318. Linderstrom-Lang, K., Hotchkiss: R. D. & Johansen, G. (1938). Nature (London), 142; 996. Matthyssens, G. E., Simons; G. & Kanarex, L. (1972). Eur. J. Biochem. 26, 449-454. Okazaki, K., Imoto, T. & Yamada; H. (1985). Anal. Biochem.
145, 87-90.
Pace, C. S. & Barret, A. J. (1984). Biochem.
J. 219, 411-
417.
Pace, C. N. & Vandenburg,
K. E. (1979). Biochemistivy, 18,
288-292. Piez, K. A.; Davie, E. W., Folk, J. E. & Gladner, J. A. (1961). J. Biol. Chem. 236, 2912-2916. Tanford, C., Pain, R. H. & Otchin, K. S. (1966). J. Mol. Biol. 15, 489-504.
by S. Brenner