Eur. J. Biochem. 54,453-458 (1975)
Spin-Labeling Studies of Urea-Treated Leucine Aminopeptidase Gunter LASSMANN, Werner DAMERAU, Gisela SKLENAR, Dieter SCHWARZ, Marlies FROHNE, Manfred LUDEWIG, and Horst HANSON Zentralinstitut fur Molekularbiologie der Akademie der Wissenschaften der DDR, Bereich Methodik und Theorie, and Physiologisch-Chemisches Institut der Martin-Luther-Universitat Halle (Received July 7INovember 12, 1974)
1. Leucine aminopeptidase (EC 3.4.11.1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-l-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A : B % 4: 1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) MnZ complexed with the protein is partly released producing hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A : B % 4: 1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely. +
Leucine aminopeptidase from bovine eye lens (mol. wt 326000 [l]) has been characterized as a metal enzyme by Kettmann and Hanson [2] and by Vahl and Carpenter [3] and is composed of six subunits [4]. The enzyme can be obtained as Zn-enzyme or as Zn/Mn-enzyme [5,6]. Six Zn atoms per hexamer are essential for the catalytic function, and 6 Mn atoms can be bound additionally [3]. In native Zn-enzyme 6 sulfhydryl groups are available for reaction with 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs,) or p-chloromercuribenzoate (pC1-HgBzOH) [7]. Iodoacetamide reacts with only 3 SH groups (M. Frohne, unpublished results). Depending on the content of additionally bound metal ions (Mn2+ or Mg2+)more thiol groups were found to be accessible [7,8]. Abbreviations. Electron spin resonance, ESR ;pchloromercuribenzoate, $1-HgBzOH; 5,5'-dithio-bis(2-nitrobenzoicacid), Nbs, ; 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-l-oxyl, I. Enzyme. Leucine aminopeptidase or L-leucyl-peptide hydrolase (EC 3.4.11.1).
Eur. J. Biochem. 54 (1975)
In order to extend the above-mentioned biochemical studies the spin-labeling method [9] was used to study structural properties of the environment of spin-labeled thiol groups in leucine aminopeptidase. In a preceding paper [lo] the polarity in the environment of thiol groups of leucine aminopeptidase was investigated. The present paper concerns conformational changes induced by urea as well as the influence of MnZ+on this process. MATERIALS AND METHODS Crystalline leucine aminopeptidase was prepared as described [ l l ] as Zn-enzyme or Zn/Mn-enzyme [5,6]. The Zn/Mn-enzyme used contained 0.5 Mn2+ per oligomer. After twice-repeated recrystallisation of the enzyme, stock solutions of 15 mg Zn-leucine aminopeptidase or Zn/Mn-enzyme/ml in 0.02 M Tris . HCl buffer, corresponding to 50 pM, were prepared and stored at pH 8.0 and 4 "C.
454
Spin-Labeling of Leucine Aminopeptidase
For a selective covalent labeling of thiol groups 2,2,6,6-tetramethyl-4- [2-iodoacetamidol-piperidine-loxyl (I; Synvar Assoc., U.S.A.) was used.
NH -CO-CH,I
0
,
1)-line
0
The labeling was carried out by mixing 50 pM enzyme solution (Tris . HCl buffer) with an equal volume of 800 pM solution of I in H 2 0 and incubation for 30 min at room temperature at pH 8.0. The excess unbound label was separated by dialysis in collodium membranes (Sartorius Membranfilter, Gottingen) against 0.02 M Tris . HCl buffer for 48 h at 8 “C and pH 8.0. After dialysis the volume of protein solution was reduced to the initial volume. The labeled leucine aminopeptidase showed no detectable loss of enzymic activity in comparison to the unlabeled enzyme. The enzymic activity was tested by m-leucine hydrazide (Fa. Reaclin, Leipzig) as substrate following the procedure given previously [I 11. The released hydrazine was determined photometrically as 4,4-dimethyl-benzylidene azine in hydrochloric solution at 436 nm (121. The degree of labeling was determined by comparison of the leucine aminopeptidase spectrum with a spectrum of an aqueous solution of the free label of known concentration (Fig. 1a). After double integration of both spectra with a digital computer (base line correction included [13]) the extent of labeling was determined. Altogether five different samples were used: two samples of Zn-enzyme containing 1 and 2.9 label molecules bound to the hexamer, and three Zn/Mn-enzyme samples with 0.6, 2.2 or 3.6 label molecules bound to the hexamer. Electron spin resonance (ESR) spectra were recorded at room temperature with a Varian E3spectrometer using a flat quartz cell. Treatment with urea was performed by incubation of 50 yM spin-labeled enzyme (Tris buffer, pH 8.0, room temperature) with 6.2 M1 urea for 5 min to 3 days. After 0.5, 2 and 4-h incubation with urea the rapid removal of the urea was accomplished by centrifugation of samples through small columns filled with Sephadex G-10 gel. External buffer solution of the gel was removed by centrifugation prior to use. In this way the enzyme was isolated without additional dilution. The separation of urea was controlled by
’ 8 M urea had no qualitatively different effect on the ESR spectra, so 6.2 M concentrations were preferred, assuring a more rapid dissolution of urea.
7-
7-
10G
A
0
A H-
Fig. I , ESR spectra ojspin label and spin-luheicd eti:ym’. (a) ESR spectrum of the free label (I) in aqueous solution (800 pM). (b) ESR spectrum of Zn/Mn-leucine arninopeptidase (50 pM) labeled with I at room temperature and pH 8 in Tris buffer (A and B indicate the two spectral parts). (c) Approximation of part B, using the free label solved in decaline at - 53 “C. (d) Difference spectrum representing part A, obtained after computer subtraction of spectrum (c) from spectrum (b)
measuring the refractive index (Abbe refractometer). The enzyme concentration was determined spectro= 9.8 at pH 8.0). photometrically RESULTS ESR Spectrum of Spin-Labeled Leucine Aminopeptidtzse
In order to test whether groups other than-SH react with the label I, under identical conditions a sample was labeled in which all 6 accessible-SH groups were blocked by pC1-HgBzOH before incubation with I. The resulting spectrum gave less than 2 ”/, of the initial spectrum of unblocked enzyme. It follows that about 98 ”/: of the bound label is localized at -SH groups Eur. J . Biochern. 34 (1975)
455
G. Lassmann, W. Damerau, G. Sklenar, D. Schwarz, M. Frohne, M. Ludewig, and H. Hanson
that are titratable by pC1-HgBzOH. The spin-labeled enzyme showed no detectable loss of its activity, which is consistent with the fact that blocking of 6 thiol groups by pC1-HgBzOH does not reduce the activity. Fig. 1b shows the ESR spectrum of Zn/Mn-enzyme labeled with I. The spectrum of the labeled enzyme can be explained as a superposition of two different spectra A and B. Part A has broad shoulders and belongs to the type of strong immobilization of the label [9]’. Part B has a line shape which is typical for weakly immobilized labels [9]. Electrophoresis of labeled enzyme gave no separation into different fractions according to the two observed spectra. The label attached to thiol groups is therefore localized in two different states of mobility. The relative concentrations of the label in the conformational states A and B were estimated by separation of the two spectral parts A and B from each other. The line shape of spectrum B was approximated by a dilute solution of label I in decaline at -53 “C, giving a quite similar splitting constant aisoand a similar line width (Fig. 1c). Successive increments of this spectrum were subtracted with the aid of a computer until the spectral part B was entirely removed [13]. The difference representing spectral part A is given in Fig. 1d. After double integration of each spectral part the relative concentrations of the label were estimated to be about 81 % in state A and 19% in state B. Under the mentioned conditions (see Materials and Methods) the line shape of the ESR spectra was independent of the degree of labeling, and no difference was found between the spectra of labeled Zn-enzyme and Zn/Mn-enzyme. A rotational correlation time zB = 2 ns for the label in the weakly immobilizing state B has been estimated according to Kuznetsov etal. [14].
Urea Treatment of ZnlMn-Leucine Aminopeptidase ESR spectra from labeled Zn/Mn-enzyme during treatment by 6.2 M urea at pH 8.0 are shown in Fig. 2. The following changes in the ESR spectra were observed. a) Immediately after addition of urea the distance between the outer lines of the spectrum A increases from 51 to 54 gauss (spectrum A’). At the same time another spectrum (B’) of weakly immobilized label
’
As seen in Fig. 1b the broad (-1)-line shows two peaks. Spectral part A seems to be composed of two subparts with slightly different mobility, which are not to be taken into account. Additional recent studies with a label rigidly bound to leucine aminopeptidase have shown (to be published), that in the case of label I used here a motion of the label relative to the enzyme molecule takes place. The value for rAgiven in [lo] is not applicable because the relative motion was not taken into account. Eur. J. Biochem. 54 (1975)
-
H-
Fig. 2. ESR spectra of spin-labeled ZnlMn-leucine arninopepritlasr after treatment by 6.2 M urea at d@erent incubation times. (-) Control without urea; (----) after 10-min incubation; (.. . ’ ..) after 2-h incubation; (-.-.-) after 23-h incubation
appears with an increased value of aiso (16.6 gauss instead of 15.3 gauss in state B). The width of the (-1)-line of B’ is 2.5 gauss indicating that the mobility of the label in the state B‘ is increased (zB,= 0.7 ns was estimated using an approximated free-label spectrum in glycerol). b) The concentration of the label bound in this state B’ increases with time. Fig. 3 shows a plot of the relative populations of the three states of A‘, B, and B’ as a function of the time of urea-denaturation. The concentrations of the label in each state were determined with the aid of a computer in a procedure similar to that described above. Immediately after addition of urea the relative concentrations of the label bound in the states A‘, B, and B’ were estimated to be about 86.5 %, 9 %, and 4.5 %, respectively (total concentration before addition of urea 100 %). The spectral analysis is complicated because the total spectrum slowly decreases, possibly due to a reduction of the -NO group of the label by released -SH groups of the unfolded enzyme. The half-life time of this conformational change was found to be about 0.5 h. Observable spectral changes are completed after 10 h. After 24 h concentrations of 76%, 5%, and 9 % correspond to the label in the conformations A‘, B, and B’, respectively. c) During denaturation of the Zn/Mn-enzyme by urea the ESR sextet from hydrated Mn2+ (not visible under recording conditions of Fig. 2) increases with incubation time (Fig. 4), indicating a release of proteinbound Mn2+during urea treatment Release of Mn2+
’.
The ESR spectrum of the Mn” . protein complex is too broad to be detected; only the hydrated Mn’+ is observable.
456
Spin-Labeling of Leucine Aminopeptidase
Total
A'
H-
Fig. 5. ESR spectra of spin-labeled Zn-leucine aminopeptidase ajter treatment by 6.2 M urea. (-) Control without urea; (----) after 17-day incubation 0' 0
L
5
10
15
20
Time (h)
Fig. 3. Time-depmdence of conformational changes during treatment of ZnIMn-leucine aminopeptidase by 6.2 M urea. A', B and B' refer to the concentrations of label bound in the different conformational states (B': unfolded state)
The growth of the concentration of the label in state B' exhibits the same time-dependence, but the extent of conformational change is less in the Mn-free enzyme. Even after 17 days of incubation the relative concentrations of the states A', B, and B' were about 73 %, 11 %, and 5 % respectively, giving 89 % of the initial spin concentration. Removal of Urea
Time ( h )
Fig. 4. Time-dependence of concentration of hydrated Mn2 ' released during incubation of ZnlMn-leucine aminopeptidase with 6.2 M urea. Concentration is given relative to the hydrated MnZ+concentration before urea treatment
ESR spectra, from the Zn-enzyme recorded immediately before and after separation of urea, showed in each case that the observed urea-induced spectral changes are fully reversible. Only the total amount of bound label was decreased, very probably due to reduction of the -NO group of a part of the bound label molecules possibly by -SH groups released by urea4. The same full reversibility of spectral changes was observed for Zn/Mn-leucine aminopeptidase, even after a 3-day incubation with urea (Fig. 6). Full initial enzymic activity was measured after removal of urea.
Urea Treatment of Zn-Leucine Aminopeptidase
DISCUSSION Because of the accessibility of at least 3 -SH groups (see Introduction) for the used 6-membered iodoacetamide label it is reasonable to assume that the label is localized at different kinds of -SH groups. Two spectral parts suggest two labeling locations,
Spectral changes of the labeled Zn-enzyme during denaturation by 6.2 M urea have been studied in comparison with Zn/Mn-enzyme (Fig. 5). The outside shift of the lines leading to states A', B' takes place in just the same manner as in the case of Zn/Mn-enzyme.
A competition for the - SH groups between label and cyanate spontaneously produced by degradation of urea can be excluded, because the protein was spin-labeled before the treatment by urea. Performing a control experiment without enzyme, in solutions containing 6.2 M urea and free spin label after 24 h at room temperature, no decrease of spin concentration was observed.
is observed both with labeled and unlabeled leucine aminopeptidase.
Eur. J. Biochem. 54 (1975)
G. Lassmann, W. Damerau, G. Sklenar, D. Schwarz, M. Frohne, M. Ludewig, and H. Hanson
457
H-
Fig. 6. ESR spectra after removal of ureafrom ZnlMn-leucine aminupeptidase. (a) Control without urea; (b) 72-h treatment with 6.2 M urea; (c) after removal of urea
A and B, at thiol groups forming different mobile environments of label. Two conformational states of the local environment of thiol-bound label molecules correspond with the two observed spectral parts. Structural parameters of local environments of the bound spin label, such as mobility of the label and polarity of its environment, were measured for state B. The correlation times in state B and A, respectively, show a different immobilization of the label by the environment of these thiol groups (free label in H,O: z = 0.06 ns). Evidence for conformational changes at the environment of labeled thiol groups were obtained after urea treatment (6.2 M) of the enzyme, leading to the appearance of a further weakly immobilizing conformational state B'. The significant increased polarity near the label and the enhanced mobility support the conclusion that B' represents an unfolded conformational state of the environment of thiol groups. State B' exists along with A' and B, and the spin concentration in B' increases with time of incubation in urea at the expense of that in state B or A', or both. A decision whether A' or B is converted into B' by curve-fitting is difficult because the whole spectrum is diminished due to a partial reduction of -NO groups. Beside this the spectral changes are weak and the three spectral parts are extremely superimposed. Protein-bound Mn2+ at the concentration of 0.5 Mn2+ per hexamer has no influence on the ratio of the two states A and B. Using an Zn/Mn-enzyme with increased Mn2+ content (5 Mn2+ per hexamer) Eur. J. Biochem. 54 (1975)
the extent of unfolding increases with increasing Mn2 content. Mn2+ seems to facilitate the urea-induced unfolding procedure. The release of Mn2+ out of the protein-bound complex during urea treatment shows kinetics comparable with those of the unfolding. Even after prolonged treatment with 6.2 or 8.0 M urea a considerable part of strongly immobilizing state A (or A') in leucine aminopeptidase remains uneffected. This is in contrast with urea-denaturation studies of other enzymes, e.g. a-chymotrypsin [15] and carbonic anhydrase [16], in which a complete unfolding was observed. +
We are gratefully indebted to B. Ebert for valuable discussions and to N. Klimes and C. Ziegler for technical assistance.
REFERENCES 1. Kretschmer, K. & Hanson, H. (1965) Hoppe-Seylers Z . Physiol. Chem. 340,126. 2. Kettmann, U. & Hanson, H. (1970) FEBS Lett. 10, 17. 3. Vahl, J. M. & Carpenter, F. H. (1971) Fed. Pruc. 30, No. 3, Part 11. 4. Damaschun, G., Damaschun, H., Hanson, H., Miiller, J. J. & Piirschel, H.-V. (1973) Stud. Biuphys. 35, 59. 5. Bottger, M., Fittkau, S . , Niese, S. & Hanson, H. (1968) Acta B i d . Med. Ger. 21, 143. 6. Kettmann, U. (1971) Thesis, University of Halle. 7. Frohne, M. & Hanson, H. (1969) Huppe-Seylers Z . Physiol. Chern. 350,213. 8. Frohne, M. & Hanson, H. (1973) Acta B i d . Med. Ger. 31,453. 9. McConnell, H. M. & McFarland, B. C. (1970) Q. Rev. Biuphys. 37, 91.
458 G. Lassmann, W. Damerau, G . Sklenar, D . Schwarz, M. Frohne, M. Ludewig, and H. Hanson: Spin-Labelingof Leucine Aminopeptidase 10. Lassmann, G., Ebert, B., Kuznetsov, A. N. & Damerau, W. (1973) Biochim. Biophys. Acta, 310, 298. 11. Hanson, H.. Glasser, D. & Kirschke, H. (1965) Hoppe-Scylers Z. Ph1,siol Chern. 340, 107. 12. Watt, G. W. & Crisp, J. D. (1952) Analyt. Chem. 24, 2006.
13. Schwarz, D. & Lassmann, G. (1973) Studiu Biophys. 3Y, 55. 14. Kuznetsov, A. N., Vasserman, A. N., Volkov, B. U . & Korst, N. N. (1971) Chem. Phys. Lett. 12, 103. 15. Berliner, L. J. (1972) Biochemistry, 11, 2921. 16. Mushag, P. & Coleman, J. E. (1972)J. Bid. Chem. 247,373.
G. Lassmann, W. Damerau, G. Sklenar, and D. Schwarz, Bereich Methodik und Theorie, Zentralinstitut fur Molekularbiologie der Akademie der Wissenschaften der DDR, DDR-1115 Berlin-Buch, Lindenberger Weg, German Democratic Republic M. Frohne, M. Ludewig, and H. Hanson, Physiologisch-Chemisches Institut der Martin-Luther-Universitat Halle, DDR-402 Hallel Saale, HollystraBe 1, German Democratic Republic
Eur. J. Biochem. 54 (1975)
Spin-Labeling Studies of Urea-Treated Leucine Aminopeptidase Gunter LASSMANN, Werner DAMERAU, Gisela SKLENAR, Dieter SCHWARZ, Marlies FROHNE, Manfred LUDEWIG, and Horst HANSON Zentralinstitut fur Molekularbiologie der Akademie der Wissenschaften der DDR, Bereich Methodik und Theorie, and Physiologisch-Chemisches Institut der Martin-Luther-Universitat Halle (Received July 7INovember 12, 1974)
1. Leucine aminopeptidase (EC 3.4.11.1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-l-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A : B % 4: 1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) MnZ complexed with the protein is partly released producing hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A : B % 4: 1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely. +
Leucine aminopeptidase from bovine eye lens (mol. wt 326000 [l]) has been characterized as a metal enzyme by Kettmann and Hanson [2] and by Vahl and Carpenter [3] and is composed of six subunits [4]. The enzyme can be obtained as Zn-enzyme or as Zn/Mn-enzyme [5,6]. Six Zn atoms per hexamer are essential for the catalytic function, and 6 Mn atoms can be bound additionally [3]. In native Zn-enzyme 6 sulfhydryl groups are available for reaction with 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs,) or p-chloromercuribenzoate (pC1-HgBzOH) [7]. Iodoacetamide reacts with only 3 SH groups (M. Frohne, unpublished results). Depending on the content of additionally bound metal ions (Mn2+ or Mg2+)more thiol groups were found to be accessible [7,8]. Abbreviations. Electron spin resonance, ESR ;pchloromercuribenzoate, $1-HgBzOH; 5,5'-dithio-bis(2-nitrobenzoicacid), Nbs, ; 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-l-oxyl, I. Enzyme. Leucine aminopeptidase or L-leucyl-peptide hydrolase (EC 3.4.11.1).
Eur. J. Biochem. 54 (1975)
In order to extend the above-mentioned biochemical studies the spin-labeling method [9] was used to study structural properties of the environment of spin-labeled thiol groups in leucine aminopeptidase. In a preceding paper [lo] the polarity in the environment of thiol groups of leucine aminopeptidase was investigated. The present paper concerns conformational changes induced by urea as well as the influence of MnZ+on this process. MATERIALS AND METHODS Crystalline leucine aminopeptidase was prepared as described [ l l ] as Zn-enzyme or Zn/Mn-enzyme [5,6]. The Zn/Mn-enzyme used contained 0.5 Mn2+ per oligomer. After twice-repeated recrystallisation of the enzyme, stock solutions of 15 mg Zn-leucine aminopeptidase or Zn/Mn-enzyme/ml in 0.02 M Tris . HCl buffer, corresponding to 50 pM, were prepared and stored at pH 8.0 and 4 "C.
454
Spin-Labeling of Leucine Aminopeptidase
For a selective covalent labeling of thiol groups 2,2,6,6-tetramethyl-4- [2-iodoacetamidol-piperidine-loxyl (I; Synvar Assoc., U.S.A.) was used.
NH -CO-CH,I
0
,
1)-line
0
The labeling was carried out by mixing 50 pM enzyme solution (Tris . HCl buffer) with an equal volume of 800 pM solution of I in H 2 0 and incubation for 30 min at room temperature at pH 8.0. The excess unbound label was separated by dialysis in collodium membranes (Sartorius Membranfilter, Gottingen) against 0.02 M Tris . HCl buffer for 48 h at 8 “C and pH 8.0. After dialysis the volume of protein solution was reduced to the initial volume. The labeled leucine aminopeptidase showed no detectable loss of enzymic activity in comparison to the unlabeled enzyme. The enzymic activity was tested by m-leucine hydrazide (Fa. Reaclin, Leipzig) as substrate following the procedure given previously [I 11. The released hydrazine was determined photometrically as 4,4-dimethyl-benzylidene azine in hydrochloric solution at 436 nm (121. The degree of labeling was determined by comparison of the leucine aminopeptidase spectrum with a spectrum of an aqueous solution of the free label of known concentration (Fig. 1a). After double integration of both spectra with a digital computer (base line correction included [13]) the extent of labeling was determined. Altogether five different samples were used: two samples of Zn-enzyme containing 1 and 2.9 label molecules bound to the hexamer, and three Zn/Mn-enzyme samples with 0.6, 2.2 or 3.6 label molecules bound to the hexamer. Electron spin resonance (ESR) spectra were recorded at room temperature with a Varian E3spectrometer using a flat quartz cell. Treatment with urea was performed by incubation of 50 yM spin-labeled enzyme (Tris buffer, pH 8.0, room temperature) with 6.2 M1 urea for 5 min to 3 days. After 0.5, 2 and 4-h incubation with urea the rapid removal of the urea was accomplished by centrifugation of samples through small columns filled with Sephadex G-10 gel. External buffer solution of the gel was removed by centrifugation prior to use. In this way the enzyme was isolated without additional dilution. The separation of urea was controlled by
’ 8 M urea had no qualitatively different effect on the ESR spectra, so 6.2 M concentrations were preferred, assuring a more rapid dissolution of urea.
7-
7-
10G
A
0
A H-
Fig. I , ESR spectra ojspin label and spin-luheicd eti:ym’. (a) ESR spectrum of the free label (I) in aqueous solution (800 pM). (b) ESR spectrum of Zn/Mn-leucine arninopeptidase (50 pM) labeled with I at room temperature and pH 8 in Tris buffer (A and B indicate the two spectral parts). (c) Approximation of part B, using the free label solved in decaline at - 53 “C. (d) Difference spectrum representing part A, obtained after computer subtraction of spectrum (c) from spectrum (b)
measuring the refractive index (Abbe refractometer). The enzyme concentration was determined spectro= 9.8 at pH 8.0). photometrically RESULTS ESR Spectrum of Spin-Labeled Leucine Aminopeptidtzse
In order to test whether groups other than-SH react with the label I, under identical conditions a sample was labeled in which all 6 accessible-SH groups were blocked by pC1-HgBzOH before incubation with I. The resulting spectrum gave less than 2 ”/, of the initial spectrum of unblocked enzyme. It follows that about 98 ”/: of the bound label is localized at -SH groups Eur. J . Biochern. 34 (1975)
455
G. Lassmann, W. Damerau, G. Sklenar, D. Schwarz, M. Frohne, M. Ludewig, and H. Hanson
that are titratable by pC1-HgBzOH. The spin-labeled enzyme showed no detectable loss of its activity, which is consistent with the fact that blocking of 6 thiol groups by pC1-HgBzOH does not reduce the activity. Fig. 1b shows the ESR spectrum of Zn/Mn-enzyme labeled with I. The spectrum of the labeled enzyme can be explained as a superposition of two different spectra A and B. Part A has broad shoulders and belongs to the type of strong immobilization of the label [9]’. Part B has a line shape which is typical for weakly immobilized labels [9]. Electrophoresis of labeled enzyme gave no separation into different fractions according to the two observed spectra. The label attached to thiol groups is therefore localized in two different states of mobility. The relative concentrations of the label in the conformational states A and B were estimated by separation of the two spectral parts A and B from each other. The line shape of spectrum B was approximated by a dilute solution of label I in decaline at -53 “C, giving a quite similar splitting constant aisoand a similar line width (Fig. 1c). Successive increments of this spectrum were subtracted with the aid of a computer until the spectral part B was entirely removed [13]. The difference representing spectral part A is given in Fig. 1d. After double integration of each spectral part the relative concentrations of the label were estimated to be about 81 % in state A and 19% in state B. Under the mentioned conditions (see Materials and Methods) the line shape of the ESR spectra was independent of the degree of labeling, and no difference was found between the spectra of labeled Zn-enzyme and Zn/Mn-enzyme. A rotational correlation time zB = 2 ns for the label in the weakly immobilizing state B has been estimated according to Kuznetsov etal. [14].
Urea Treatment of ZnlMn-Leucine Aminopeptidase ESR spectra from labeled Zn/Mn-enzyme during treatment by 6.2 M urea at pH 8.0 are shown in Fig. 2. The following changes in the ESR spectra were observed. a) Immediately after addition of urea the distance between the outer lines of the spectrum A increases from 51 to 54 gauss (spectrum A’). At the same time another spectrum (B’) of weakly immobilized label
’
As seen in Fig. 1b the broad (-1)-line shows two peaks. Spectral part A seems to be composed of two subparts with slightly different mobility, which are not to be taken into account. Additional recent studies with a label rigidly bound to leucine aminopeptidase have shown (to be published), that in the case of label I used here a motion of the label relative to the enzyme molecule takes place. The value for rAgiven in [lo] is not applicable because the relative motion was not taken into account. Eur. J. Biochem. 54 (1975)
-
H-
Fig. 2. ESR spectra of spin-labeled ZnlMn-leucine arninopepritlasr after treatment by 6.2 M urea at d@erent incubation times. (-) Control without urea; (----) after 10-min incubation; (.. . ’ ..) after 2-h incubation; (-.-.-) after 23-h incubation
appears with an increased value of aiso (16.6 gauss instead of 15.3 gauss in state B). The width of the (-1)-line of B’ is 2.5 gauss indicating that the mobility of the label in the state B‘ is increased (zB,= 0.7 ns was estimated using an approximated free-label spectrum in glycerol). b) The concentration of the label bound in this state B’ increases with time. Fig. 3 shows a plot of the relative populations of the three states of A‘, B, and B’ as a function of the time of urea-denaturation. The concentrations of the label in each state were determined with the aid of a computer in a procedure similar to that described above. Immediately after addition of urea the relative concentrations of the label bound in the states A‘, B, and B’ were estimated to be about 86.5 %, 9 %, and 4.5 %, respectively (total concentration before addition of urea 100 %). The spectral analysis is complicated because the total spectrum slowly decreases, possibly due to a reduction of the -NO group of the label by released -SH groups of the unfolded enzyme. The half-life time of this conformational change was found to be about 0.5 h. Observable spectral changes are completed after 10 h. After 24 h concentrations of 76%, 5%, and 9 % correspond to the label in the conformations A‘, B, and B’, respectively. c) During denaturation of the Zn/Mn-enzyme by urea the ESR sextet from hydrated Mn2+ (not visible under recording conditions of Fig. 2) increases with incubation time (Fig. 4), indicating a release of proteinbound Mn2+during urea treatment Release of Mn2+
’.
The ESR spectrum of the Mn” . protein complex is too broad to be detected; only the hydrated Mn’+ is observable.
456
Spin-Labeling of Leucine Aminopeptidase
Total
A'
H-
Fig. 5. ESR spectra of spin-labeled Zn-leucine aminopeptidase ajter treatment by 6.2 M urea. (-) Control without urea; (----) after 17-day incubation 0' 0
L
5
10
15
20
Time (h)
Fig. 3. Time-depmdence of conformational changes during treatment of ZnIMn-leucine aminopeptidase by 6.2 M urea. A', B and B' refer to the concentrations of label bound in the different conformational states (B': unfolded state)
The growth of the concentration of the label in state B' exhibits the same time-dependence, but the extent of conformational change is less in the Mn-free enzyme. Even after 17 days of incubation the relative concentrations of the states A', B, and B' were about 73 %, 11 %, and 5 % respectively, giving 89 % of the initial spin concentration. Removal of Urea
Time ( h )
Fig. 4. Time-dependence of concentration of hydrated Mn2 ' released during incubation of ZnlMn-leucine aminopeptidase with 6.2 M urea. Concentration is given relative to the hydrated MnZ+concentration before urea treatment
ESR spectra, from the Zn-enzyme recorded immediately before and after separation of urea, showed in each case that the observed urea-induced spectral changes are fully reversible. Only the total amount of bound label was decreased, very probably due to reduction of the -NO group of a part of the bound label molecules possibly by -SH groups released by urea4. The same full reversibility of spectral changes was observed for Zn/Mn-leucine aminopeptidase, even after a 3-day incubation with urea (Fig. 6). Full initial enzymic activity was measured after removal of urea.
Urea Treatment of Zn-Leucine Aminopeptidase
DISCUSSION Because of the accessibility of at least 3 -SH groups (see Introduction) for the used 6-membered iodoacetamide label it is reasonable to assume that the label is localized at different kinds of -SH groups. Two spectral parts suggest two labeling locations,
Spectral changes of the labeled Zn-enzyme during denaturation by 6.2 M urea have been studied in comparison with Zn/Mn-enzyme (Fig. 5). The outside shift of the lines leading to states A', B' takes place in just the same manner as in the case of Zn/Mn-enzyme.
A competition for the - SH groups between label and cyanate spontaneously produced by degradation of urea can be excluded, because the protein was spin-labeled before the treatment by urea. Performing a control experiment without enzyme, in solutions containing 6.2 M urea and free spin label after 24 h at room temperature, no decrease of spin concentration was observed.
is observed both with labeled and unlabeled leucine aminopeptidase.
Eur. J. Biochem. 54 (1975)
G. Lassmann, W. Damerau, G. Sklenar, D. Schwarz, M. Frohne, M. Ludewig, and H. Hanson
457
H-
Fig. 6. ESR spectra after removal of ureafrom ZnlMn-leucine aminupeptidase. (a) Control without urea; (b) 72-h treatment with 6.2 M urea; (c) after removal of urea
A and B, at thiol groups forming different mobile environments of label. Two conformational states of the local environment of thiol-bound label molecules correspond with the two observed spectral parts. Structural parameters of local environments of the bound spin label, such as mobility of the label and polarity of its environment, were measured for state B. The correlation times in state B and A, respectively, show a different immobilization of the label by the environment of these thiol groups (free label in H,O: z = 0.06 ns). Evidence for conformational changes at the environment of labeled thiol groups were obtained after urea treatment (6.2 M) of the enzyme, leading to the appearance of a further weakly immobilizing conformational state B'. The significant increased polarity near the label and the enhanced mobility support the conclusion that B' represents an unfolded conformational state of the environment of thiol groups. State B' exists along with A' and B, and the spin concentration in B' increases with time of incubation in urea at the expense of that in state B or A', or both. A decision whether A' or B is converted into B' by curve-fitting is difficult because the whole spectrum is diminished due to a partial reduction of -NO groups. Beside this the spectral changes are weak and the three spectral parts are extremely superimposed. Protein-bound Mn2+ at the concentration of 0.5 Mn2+ per hexamer has no influence on the ratio of the two states A and B. Using an Zn/Mn-enzyme with increased Mn2+ content (5 Mn2+ per hexamer) Eur. J. Biochem. 54 (1975)
the extent of unfolding increases with increasing Mn2 content. Mn2+ seems to facilitate the urea-induced unfolding procedure. The release of Mn2+ out of the protein-bound complex during urea treatment shows kinetics comparable with those of the unfolding. Even after prolonged treatment with 6.2 or 8.0 M urea a considerable part of strongly immobilizing state A (or A') in leucine aminopeptidase remains uneffected. This is in contrast with urea-denaturation studies of other enzymes, e.g. a-chymotrypsin [15] and carbonic anhydrase [16], in which a complete unfolding was observed. +
We are gratefully indebted to B. Ebert for valuable discussions and to N. Klimes and C. Ziegler for technical assistance.
REFERENCES 1. Kretschmer, K. & Hanson, H. (1965) Hoppe-Seylers Z . Physiol. Chem. 340,126. 2. Kettmann, U. & Hanson, H. (1970) FEBS Lett. 10, 17. 3. Vahl, J. M. & Carpenter, F. H. (1971) Fed. Pruc. 30, No. 3, Part 11. 4. Damaschun, G., Damaschun, H., Hanson, H., Miiller, J. J. & Piirschel, H.-V. (1973) Stud. Biuphys. 35, 59. 5. Bottger, M., Fittkau, S . , Niese, S. & Hanson, H. (1968) Acta B i d . Med. Ger. 21, 143. 6. Kettmann, U. (1971) Thesis, University of Halle. 7. Frohne, M. & Hanson, H. (1969) Huppe-Seylers Z . Physiol. Chern. 350,213. 8. Frohne, M. & Hanson, H. (1973) Acta B i d . Med. Ger. 31,453. 9. McConnell, H. M. & McFarland, B. C. (1970) Q. Rev. Biuphys. 37, 91.
458 G. Lassmann, W. Damerau, G . Sklenar, D . Schwarz, M. Frohne, M. Ludewig, and H. Hanson: Spin-Labelingof Leucine Aminopeptidase 10. Lassmann, G., Ebert, B., Kuznetsov, A. N. & Damerau, W. (1973) Biochim. Biophys. Acta, 310, 298. 11. Hanson, H.. Glasser, D. & Kirschke, H. (1965) Hoppe-Scylers Z. Ph1,siol Chern. 340, 107. 12. Watt, G. W. & Crisp, J. D. (1952) Analyt. Chem. 24, 2006.
13. Schwarz, D. & Lassmann, G. (1973) Studiu Biophys. 3Y, 55. 14. Kuznetsov, A. N., Vasserman, A. N., Volkov, B. U . & Korst, N. N. (1971) Chem. Phys. Lett. 12, 103. 15. Berliner, L. J. (1972) Biochemistry, 11, 2921. 16. Mushag, P. & Coleman, J. E. (1972)J. Bid. Chem. 247,373.
G. Lassmann, W. Damerau, G. Sklenar, and D. Schwarz, Bereich Methodik und Theorie, Zentralinstitut fur Molekularbiologie der Akademie der Wissenschaften der DDR, DDR-1115 Berlin-Buch, Lindenberger Weg, German Democratic Republic M. Frohne, M. Ludewig, and H. Hanson, Physiologisch-Chemisches Institut der Martin-Luther-Universitat Halle, DDR-402 Hallel Saale, HollystraBe 1, German Democratic Republic
Eur. J. Biochem. 54 (1975)
