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Raman resonance studies of functional fragments of helix pomatia βC-haemocyanin
Raman Resonance Studies of Functional Fragments of Helixpomatia & -Haemocyanin Constant Gielens, Guido Maes, Th&&e Zeegers-Huyskens, and
Rene Lontie Laboratorium voor Biochemie. Katholieke Universiteit re Leuven. and Loborarorium voor Fysicochemie en Straiingschemie, Katholieke Universireit te Leuven (GM and IZH)
ABSTRACT Resonance Raman spectra were recorded for tenth molecules of fro-haemocyanin of Helixpomati, for a limited tryptic hydrolysate, and for the fwe isolated fragments. On excitation at 514.5 nm the intensity of the peroxide stretching vibration was preserved in the fragments. Domain h showed a frequency of 741 cm-l in contrast to 745-747 cm-1 for the other fragments. On excitation at 457.9 nm the intensity of the Cu-N vibration at 270 cm-l was lowered on partial txypsinolysis, suggesting a less symmetrical arrangement of the ligands of Cu in the fragments.
INTRODUCTION The haemocyanin of the mollusc Helix porn&& with M, 9 X lo6 [l] consists of an o-component, which dissociates into half molecules in the pH-stability region in the presence of I M NaCl or KCI, and of a &component, which does not dissociate under those conditions [2] _ Part of the &baemocyanin crystallizes upon dialysis at the isoelectric point @H 5.3) at low ionic strength (10 mM) [3] . This crystalline fi-haemocyardn @c-Hc) consists of 20 identical polypeptide chains, each folded in 8 oxygenbinding domains (a-h) of average M, 55,000 [4] _ Each domain contains two Cu and binds one dioxygen. By the action of trypsin on tenth molecuies (M,. 9 X 105) five proteolytic fragments were obtained [5], which were located in the polypeptide chain [6] : TlA(a-c), Address reprint
m(d),
TlC(ef),
T2(g), TlB(h).
requeststo: R. Lo&e, Laboratorium voor Biochemie, Dekenstraat 6, B-3000 Leuven, Belgium.
Journal oflnogonic Btiehemktry 13,4147 (1980) 0 F&evier North Holland, Inc., 1980 52 Vanderbilt Ave.. New York, New York 10017
0i62-0134/80/050041-07502.~
Constant Gielens et al.
42
These fmgrnents remained fulIy functional as indicated by the preservation of the absorbance at 346 ML Their circular dichroic spectrum indicated the presence of two classesof copper groups according to their positive extremum at 455 or at 500 nm [S] . Raman resonance peaks at 742 and 282 cm-l have been observed with oxyhaemocyanin of the arthropod &rcer magisrer [7] _ The peak at 742 cm-l is in resonance with the absorption bands at 575 and at 505 run and has been attributed to a peroxide stretching vibration, as it shifts to 704 cm-l on substitutingl*On for 1602. The peak at 282 cm-l, in resonance with the absorption band at 340 nm, has been assignedto a Cu-N(imidazole) vibration [8] _ With the oxyhaemocyanin of the mollusc Busycon cen~Z~c&~~furn frequencies have been observed at 749 and 267 cm-l [8]. On excitation at 351-l or 363.8 nm, bands were observed at 287 and 266 cm-1 for theoxyhaemocyanin of the arthropod LimuIus polyphemus and of B. cadiculotum, respectively, together with severalweaker bands [9] _ The fractions of L. polyphemus haemocyanin, obtained by chromatography on DEA.E-celtulose,were also compared on excitation at 351-l and 363.8 nm and showed small differences in the distribution of the resonance Peaks PI In thismanner we compared the wavenumber and the relative Raman intensity of the peroxide stretching vibration and of the Cu-N vibration of tenth molecules of Bo-haemocyanin of JL pomahb, of the limited tryptic hydrolysate, and of the five isolated tryptic fragments in view of the observed differences in the circular dichroic spectrum of the domains [S] _ EXPERIMENTAL Materials
Total haemocyanin of H. pomati was prepared according to Heirwegh et al. [2] and solubihzed in 0.1 M acetate buffer, pH 5.7. &Haemocyanin was separated by dialysis of the total preparation at 4°C against 10 mM acetate buffer, pH 5.3. It was dissolved in phosphate buffer, pH 6.5, IO.1 M, 0.02% NaNa, and recrystallized. After solubilization in the phosphate buffer of pH 6.5 at a concentration of 25 mg/ml the solution was GItered through a Millipore filter type HAWP in a 90-mm Hi-Fhrx cell (Millipore Carp_, Bedford, MA)_ Tenth molecules of &-haemocyanin were obtained by dialysis against borate-HCI buffer, pH 8.2, IO.1 M They were split into fragmentsby the action of bovine pancreatic trypsin (Batch 261-2), a gift from Novo Industri A/S (Copenhagen, Denmark)_ After an incubation for 20 min at room temperature with an enzyme to substrate ratio of l/400 (w/w) the hydrolysis was stopped by the addition of hen egg-white trypsin inhibitor (Boehringer, Mannheim, Germany) in a molar ratio of 2 with respect to trypsin. The tryptic fi-agurentswere separated by chromatography on DEABSepharose CL&B (Pharmacia Fme Chemicals, Uppsala, Sweden) using a linear NaCl gradient (0.1-0.6 M) in Tris-HClbuffer, pH 8.2JO.05 M. The column (48.5 X 3.6 cm) was loaded with approximately 500 mg hydrolysate and eluted at 90 ml/h in fractions of 10 ml. The fragments eluting in the order T3, T2, Tl A, TlB, and TIC were concentrated to about 20 mgjml on a DiafIo PM 10 membrane (Amicon Corp., Lexington, MA), mounted in the Millipore go-mm Hi-Fhrx cell. They, as well as the intact tenths and the total hydrolysate, were regenerated by the addition of HaOs in a molar ratio of 10 with respect to Crr [lo] _ After a reaction time of 1 hr the solutions were extensively
Raman Resonance of Haemocyanin
Fragments
43
dialysed against borate-HCl buffer, pH 8.2, IO.1 M. The purity of the fragments was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Methods The Raman spectra were recorded on a Coderg T800 spectrophotometer using slit widths ranging from 6 to 8 cm- l. The spectrophotometer was equipped with a Spectra Physics argon ion laser model 164 emitting a power of 800 mW at 514.5 mn and of 200 mW at 457.9 nm. The frequencies were calibrated at v. and vI (CCl,) and were affected with an uncertainty of 9.5 cm-l. The intensities of the Raman lines were determined by planimetry using the borate peak at 873 cm-1 as an internal standard. After irradiation of the samples a decrease of the absorbance at 346 nm of 2% was observed, except for domain d, where the loss amounted to 5%. The absorption spectrum of domains g and h was recorded at room temperature in a Perkin Elmer 554 spectrophotometer at protein concentrations of 19.4 and 16.0 mg/ml, respectively. The copper content of the samples was determined in a Perkin Elmer 372 atomic absorption spectrophotometer at 324.7 run. RESULTS AND DISCUSSION The Rarnan resonance spectra were recorded in borafe-HCl buffer, pH 8.2, IO.1 M for tenth molecules of &haemocyanin in order to reduce Rayleigh scattering. They were also taken in the same buffer for the total tryptic hydrolysate and the separated fragments. The Raman intensities were normalized for the absorbance at 346 mn characteristic for the active copper groups_ The Peroxide Stretching Vibration
The wavenumber and the relative Raman intensity of the peroxide stretching vibration normalized for the absorbance at 346 nm are presented in Table 1. No significant
TABLE 1. Wavenumber of the ~~(0-0) Vibration, Intensity Relative to the Borate Peak (&), and Normalized for the Absorbance at 346 nm (1JAa4enm). on Excitation at 5 14.5 run Preparation
g/l
sc-IIc Tryptic hydrolysate Fragment u-c Domain d Fragmentef Domain g Domain h
19.1
745
0.605 O.Ol=
5.05
12-4 21.5 20.7 17.3 21.4 18.6
745 745 747 745 746 741
0.36f 0.03 056 i 0.03 0.63f 0.02 0.58* 0.04 0.63k 0.04 0.60+ 0.05
4.8* 4.31 4.14 4.97 4.59 4.99
=Standarddeviation(n=55).
Av(crr+)
44
Constant
Gielens et al.
56
borate
741
900 FIGURE
1. Resonance Raman
850
800
750
700
AWcm -1 I spec?rum showing the borate reference peak at 873 cm-1 and the
v@-O) band of (a) domain g, 21.4 mg/ml, 0.74 mM Co, and (b) domain h, 18.6 mg/ml, 0.61 mhf Cu. using the 5145 run line of an argon ion laser- The slit width is 8 cm-l, and the scanning speed is 10 cm-1 mir-1.
FIGURE 2- The molar absorption coefficient ECU, expressed per mole of Cu, in the visiile region of domaiusg andh_
1000 -
h
n-
Eoo
I
I
500
600
1
nm
700
Raman Resonance of Haemocyanin
Fragments
45
intensity difference was noted between &+aemocyanin, the tryptic hydrolysate, and the five fragments. Small differences were observed for the wavenumber of this vibration, the lowest value being noted at 741 cm-1 for domain h (Figure 1). The absorption band in the 550-580 nm region involves a charge transfer O,a- + Cu(II) [7,8], which strengthens the O-O bond. The low value observed for domain h suggests that the charge transfer in the ground state is a little less pronounced in domain h than in the other fragments. This is in agreement with the difference in the absorption spectrum which shows a maximum at 550 run for domain h but at 570 nm for the other fragments, as exemplified by domain g (Figure 2). As a rule, the lower the charge transfer, the higher the frequency of the charge transfer band [l l] . From the wavelength dependence of ye (electronic state responsible for resonance) the FA factor of Albrecht and HutIey [ 121 predicts an intensity enhancement of the peroxide vibration on excitation at 514.5 nm five times larger for domain h than for the other fragments. The observed ratio of only 1.10 can be explained by a rather high damping constant, as suggested by the large bandwidth (x4000 cm-l). On deducing the FA factor, damping near the absorption maximum was neglected [13]. The same explanation was offered for C. magister haemocyanin by Loehr et al. [7]. They performed measurements at different excitation wavelengths and found that the calculated intensity enhancement of the 742 cm-1 band at 514.5 nm relative to 488.0 run was about twice the observed vahre. The Cu-N Stretching Vibration The relative intensity normalized for the absorbance at 346 mn of the (C&N) stretching vibration is listed in Table 2. The band was observed at 270 cm-l for tenth molecules of &&aemocyanin, the partial tryptic hydrolysate, and the five fragments, indicating that the wavenumber of this vibration is not very sensitive to small differences in the.0a2--f Cu(II) charge transfer_ Ia L&I& complexes (L = 1-methylimidazole, pyridine; X = CI, Br) the Cu-N vibration is actually quite insensitive to the electronegativity of the X atom [14], i.e., to weak variations of the charge on the 0.1 atom. A striking decrease of the intensity of the Cu-N band was observed for the tryptic hydrolysate and for the five isolated fragments in comparison with the tenth molecules. The spectra of domains g and h are compared with the spectrum of tenth moleTABLE 2. Relative Intensity (In) and Intensity Normalized for the Absorbance at 346 nm (1n/Aa4anm) of the Cu-N Stretching Vibration at 270 cm-1 on Excitation at 457.9 nm Preparation
Bc-Hc
8/l
k
_
19.1
1.33 f 0.14=
12.4 21.5 20.7 17.3 21.4 18.6
0.36 f 0.49 * 0.61= 0.41 * 0.58 + 0~53 f
IRb
346nm
11.13
Tryptic hydrolysate Fragment a-c Domain d Fragment ef Domain g Domain h 4 Standard deviation (rz = 5).
0.02 0.05 0.01 0.03 0.04 0.08
4.86 3.75 3.99 3.53 4.19 4-43
Constant Gielens et aL
borate
b
C
I
900
850
I
I
250
300
Avtcm-'I and the v(Cu-N) band at 270 cm-1 of (a) domaing, 21.4 mghl, 0.74 mbf Cu, (IJ) domain h, 18.6 m&l, 0.61 m&i Cu. and (c) tenth molecules of ~c-haemocyanin, 19.0 mghnl, 0.69 mM Cu, using the 457.9 nm line of an argon ion laser. The slit width is 6 cm-1 and the scamdug speed is 10 an-1 FIGURE
3. Resonance Raman qxc?mm
showing the borate reference peak at 873 cm-l
Inin=?. c&s of &&aemocyanin (Figure 3). The important lowering of the intensity of this band suggests a m$ifkaticn of the orientation of the ligandsof copper, likely imidazole groups, which probably becomes more asymmetric.
CONCLUSIONS The results of this study co&m that the tryptic fragments contain well-preserved active sites, as shown by the preservation of the ~$0-0) v&ration. The orientation of the ligands of Cu may somewhat differ in the fragments and in the tenth molecules of &-haemocyanin, as suggested by the decrease of the intensity of the v(C!u-N) vibration, tie are gnGq2 to professor D- ShCver, Evanston, IL. for he&@ debted to the N&naal Fends voor Wetemdtappelijk Ondenoek
disacszions. The authors are infor a f&w&p of Qangetteki
Raman Resonance of Haemocyanin
Fragments
47
Navorsw” [GM), to the Bel,#an Government [Convention No. 76.81l.II.4). and to the Fonds voor CNIectief Fundomenteel Ondersoek. Brussels, Belgium. for research grants (Contract No. 2.0016.76).
REFERENCES 1. E. J. Wood, W. H. Bannister,C. J. Oliver, R. Lontie, and R. Witters, Camp. Biochem. Phytiol. 4OB, 19-24 (1971). 2 K_ Heirwe&, H. Borginon,and R_ Lo&e, B.i&him_ Biophyz Acta 48,.517-526 (1961). 3. C. Gielens,G. Pr&ux, and R. Lontie, Arch_ Internat_ PhysroL Biochiin_ 81,182-183 (1973). 4. C. Gielens, G. Pr&aux,and R. Lontie, in Stmcture and Function of Haemocyanin, J. V. Bannister,Ed. Springer-Verlag,Berlin, 1977, pp_ 85-94. 5. C. GieIens,G. Pre’aux,and R. Lontie, Eur. J. Biochem. 60,271-280 (1975). 6. G. Pr&ux, C. Gielens, L. Verschueren,and R. Lontie, FEBS Special Meeting on Enzymes, Dubrovnik, 1979, S3-86.Sve&I&a nakIadaLiber, Zagreb, 1979. 7. J. S. Loehr, T. B. Freedman, and T. M. Loehr, Biochem. Biophys Res Commun 56, SlO515 (1974). 8. T. B. Freedman,J. S. Loch;, and T. M. Loehr, J. Am. Chem Sot. 98,2809-2815 (1976). 9. J. A. Larrabee,T. G. Spiro, N. S. Ferris,W. H. Woodruff, W. A. Maltese, and M. S. Kerr, J. Am them_ Socr 99,1979-1980 (1977). 10. K. Heirwegh,V. Blatoqand R. Lontie, Arch. Internat. PhysioZ. Biochim. 73,149-150 (1965). 11. R. S. Mulliken and W. B. Person,Moienrlm Comphzxes, Wiley Interscience,Chichester,1969. 12_ A_ C!_AIbrechtand M_ C. HutIey,Z them Phyz 55,4438-3 (1971)_ 13. L- A. Natie, R. W. Pastor, J. C. Dabrowiak, and W. H_ Woodruff, J. Anr Chem. Sot. 98, 8007-8014 (1976). 14. J. R. Ferraro, LOP/-Frt?qUenCy Vibrations of Inorganic and Coo&nation Compounds Plenum Press,New York, 1971.
Received December I, 1979; revised January 2,198O
Rene Lontie Laboratorium voor Biochemie. Katholieke Universiteit re Leuven. and Loborarorium voor Fysicochemie en Straiingschemie, Katholieke Universireit te Leuven (GM and IZH)
ABSTRACT Resonance Raman spectra were recorded for tenth molecules of fro-haemocyanin of Helixpomati, for a limited tryptic hydrolysate, and for the fwe isolated fragments. On excitation at 514.5 nm the intensity of the peroxide stretching vibration was preserved in the fragments. Domain h showed a frequency of 741 cm-l in contrast to 745-747 cm-1 for the other fragments. On excitation at 457.9 nm the intensity of the Cu-N vibration at 270 cm-l was lowered on partial txypsinolysis, suggesting a less symmetrical arrangement of the ligands of Cu in the fragments.
INTRODUCTION The haemocyanin of the mollusc Helix porn&& with M, 9 X lo6 [l] consists of an o-component, which dissociates into half molecules in the pH-stability region in the presence of I M NaCl or KCI, and of a &component, which does not dissociate under those conditions [2] _ Part of the &baemocyanin crystallizes upon dialysis at the isoelectric point @H 5.3) at low ionic strength (10 mM) [3] . This crystalline fi-haemocyardn @c-Hc) consists of 20 identical polypeptide chains, each folded in 8 oxygenbinding domains (a-h) of average M, 55,000 [4] _ Each domain contains two Cu and binds one dioxygen. By the action of trypsin on tenth molecuies (M,. 9 X 105) five proteolytic fragments were obtained [5], which were located in the polypeptide chain [6] : TlA(a-c), Address reprint
m(d),
TlC(ef),
T2(g), TlB(h).
requeststo: R. Lo&e, Laboratorium voor Biochemie, Dekenstraat 6, B-3000 Leuven, Belgium.
Journal oflnogonic Btiehemktry 13,4147 (1980) 0 F&evier North Holland, Inc., 1980 52 Vanderbilt Ave.. New York, New York 10017
0i62-0134/80/050041-07502.~
Constant Gielens et al.
42
These fmgrnents remained fulIy functional as indicated by the preservation of the absorbance at 346 ML Their circular dichroic spectrum indicated the presence of two classesof copper groups according to their positive extremum at 455 or at 500 nm [S] . Raman resonance peaks at 742 and 282 cm-l have been observed with oxyhaemocyanin of the arthropod &rcer magisrer [7] _ The peak at 742 cm-l is in resonance with the absorption bands at 575 and at 505 run and has been attributed to a peroxide stretching vibration, as it shifts to 704 cm-l on substitutingl*On for 1602. The peak at 282 cm-l, in resonance with the absorption band at 340 nm, has been assignedto a Cu-N(imidazole) vibration [8] _ With the oxyhaemocyanin of the mollusc Busycon cen~Z~c&~~furn frequencies have been observed at 749 and 267 cm-l [8]. On excitation at 351-l or 363.8 nm, bands were observed at 287 and 266 cm-1 for theoxyhaemocyanin of the arthropod LimuIus polyphemus and of B. cadiculotum, respectively, together with severalweaker bands [9] _ The fractions of L. polyphemus haemocyanin, obtained by chromatography on DEA.E-celtulose,were also compared on excitation at 351-l and 363.8 nm and showed small differences in the distribution of the resonance Peaks PI In thismanner we compared the wavenumber and the relative Raman intensity of the peroxide stretching vibration and of the Cu-N vibration of tenth molecules of Bo-haemocyanin of JL pomahb, of the limited tryptic hydrolysate, and of the five isolated tryptic fragments in view of the observed differences in the circular dichroic spectrum of the domains [S] _ EXPERIMENTAL Materials
Total haemocyanin of H. pomati was prepared according to Heirwegh et al. [2] and solubihzed in 0.1 M acetate buffer, pH 5.7. &Haemocyanin was separated by dialysis of the total preparation at 4°C against 10 mM acetate buffer, pH 5.3. It was dissolved in phosphate buffer, pH 6.5, IO.1 M, 0.02% NaNa, and recrystallized. After solubilization in the phosphate buffer of pH 6.5 at a concentration of 25 mg/ml the solution was GItered through a Millipore filter type HAWP in a 90-mm Hi-Fhrx cell (Millipore Carp_, Bedford, MA)_ Tenth molecules of &-haemocyanin were obtained by dialysis against borate-HCI buffer, pH 8.2, IO.1 M They were split into fragmentsby the action of bovine pancreatic trypsin (Batch 261-2), a gift from Novo Industri A/S (Copenhagen, Denmark)_ After an incubation for 20 min at room temperature with an enzyme to substrate ratio of l/400 (w/w) the hydrolysis was stopped by the addition of hen egg-white trypsin inhibitor (Boehringer, Mannheim, Germany) in a molar ratio of 2 with respect to trypsin. The tryptic fi-agurentswere separated by chromatography on DEABSepharose CL&B (Pharmacia Fme Chemicals, Uppsala, Sweden) using a linear NaCl gradient (0.1-0.6 M) in Tris-HClbuffer, pH 8.2JO.05 M. The column (48.5 X 3.6 cm) was loaded with approximately 500 mg hydrolysate and eluted at 90 ml/h in fractions of 10 ml. The fragments eluting in the order T3, T2, Tl A, TlB, and TIC were concentrated to about 20 mgjml on a DiafIo PM 10 membrane (Amicon Corp., Lexington, MA), mounted in the Millipore go-mm Hi-Fhrx cell. They, as well as the intact tenths and the total hydrolysate, were regenerated by the addition of HaOs in a molar ratio of 10 with respect to Crr [lo] _ After a reaction time of 1 hr the solutions were extensively
Raman Resonance of Haemocyanin
Fragments
43
dialysed against borate-HCl buffer, pH 8.2, IO.1 M. The purity of the fragments was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Methods The Raman spectra were recorded on a Coderg T800 spectrophotometer using slit widths ranging from 6 to 8 cm- l. The spectrophotometer was equipped with a Spectra Physics argon ion laser model 164 emitting a power of 800 mW at 514.5 mn and of 200 mW at 457.9 nm. The frequencies were calibrated at v. and vI (CCl,) and were affected with an uncertainty of 9.5 cm-l. The intensities of the Raman lines were determined by planimetry using the borate peak at 873 cm-1 as an internal standard. After irradiation of the samples a decrease of the absorbance at 346 nm of 2% was observed, except for domain d, where the loss amounted to 5%. The absorption spectrum of domains g and h was recorded at room temperature in a Perkin Elmer 554 spectrophotometer at protein concentrations of 19.4 and 16.0 mg/ml, respectively. The copper content of the samples was determined in a Perkin Elmer 372 atomic absorption spectrophotometer at 324.7 run. RESULTS AND DISCUSSION The Rarnan resonance spectra were recorded in borafe-HCl buffer, pH 8.2, IO.1 M for tenth molecules of &haemocyanin in order to reduce Rayleigh scattering. They were also taken in the same buffer for the total tryptic hydrolysate and the separated fragments. The Raman intensities were normalized for the absorbance at 346 mn characteristic for the active copper groups_ The Peroxide Stretching Vibration
The wavenumber and the relative Raman intensity of the peroxide stretching vibration normalized for the absorbance at 346 nm are presented in Table 1. No significant
TABLE 1. Wavenumber of the ~~(0-0) Vibration, Intensity Relative to the Borate Peak (&), and Normalized for the Absorbance at 346 nm (1JAa4enm). on Excitation at 5 14.5 run Preparation
g/l
sc-IIc Tryptic hydrolysate Fragment u-c Domain d Fragmentef Domain g Domain h
19.1
745
0.605 O.Ol=
5.05
12-4 21.5 20.7 17.3 21.4 18.6
745 745 747 745 746 741
0.36f 0.03 056 i 0.03 0.63f 0.02 0.58* 0.04 0.63k 0.04 0.60+ 0.05
4.8* 4.31 4.14 4.97 4.59 4.99
=Standarddeviation(n=55).
Av(crr+)
44
Constant
Gielens et al.
56
borate
741
900 FIGURE
1. Resonance Raman
850
800
750
700
AWcm -1 I spec?rum showing the borate reference peak at 873 cm-1 and the
v@-O) band of (a) domain g, 21.4 mg/ml, 0.74 mM Co, and (b) domain h, 18.6 mg/ml, 0.61 mhf Cu. using the 5145 run line of an argon ion laser- The slit width is 8 cm-l, and the scanning speed is 10 cm-1 mir-1.
FIGURE 2- The molar absorption coefficient ECU, expressed per mole of Cu, in the visiile region of domaiusg andh_
1000 -
h
n-
Eoo
I
I
500
600
1
nm
700
Raman Resonance of Haemocyanin
Fragments
45
intensity difference was noted between &+aemocyanin, the tryptic hydrolysate, and the five fragments. Small differences were observed for the wavenumber of this vibration, the lowest value being noted at 741 cm-1 for domain h (Figure 1). The absorption band in the 550-580 nm region involves a charge transfer O,a- + Cu(II) [7,8], which strengthens the O-O bond. The low value observed for domain h suggests that the charge transfer in the ground state is a little less pronounced in domain h than in the other fragments. This is in agreement with the difference in the absorption spectrum which shows a maximum at 550 run for domain h but at 570 nm for the other fragments, as exemplified by domain g (Figure 2). As a rule, the lower the charge transfer, the higher the frequency of the charge transfer band [l l] . From the wavelength dependence of ye (electronic state responsible for resonance) the FA factor of Albrecht and HutIey [ 121 predicts an intensity enhancement of the peroxide vibration on excitation at 514.5 nm five times larger for domain h than for the other fragments. The observed ratio of only 1.10 can be explained by a rather high damping constant, as suggested by the large bandwidth (x4000 cm-l). On deducing the FA factor, damping near the absorption maximum was neglected [13]. The same explanation was offered for C. magister haemocyanin by Loehr et al. [7]. They performed measurements at different excitation wavelengths and found that the calculated intensity enhancement of the 742 cm-1 band at 514.5 nm relative to 488.0 run was about twice the observed vahre. The Cu-N Stretching Vibration The relative intensity normalized for the absorbance at 346 mn of the (C&N) stretching vibration is listed in Table 2. The band was observed at 270 cm-l for tenth molecules of &&aemocyanin, the partial tryptic hydrolysate, and the five fragments, indicating that the wavenumber of this vibration is not very sensitive to small differences in the.0a2--f Cu(II) charge transfer_ Ia L&I& complexes (L = 1-methylimidazole, pyridine; X = CI, Br) the Cu-N vibration is actually quite insensitive to the electronegativity of the X atom [14], i.e., to weak variations of the charge on the 0.1 atom. A striking decrease of the intensity of the Cu-N band was observed for the tryptic hydrolysate and for the five isolated fragments in comparison with the tenth molecules. The spectra of domains g and h are compared with the spectrum of tenth moleTABLE 2. Relative Intensity (In) and Intensity Normalized for the Absorbance at 346 nm (1n/Aa4anm) of the Cu-N Stretching Vibration at 270 cm-1 on Excitation at 457.9 nm Preparation
Bc-Hc
8/l
k
_
19.1
1.33 f 0.14=
12.4 21.5 20.7 17.3 21.4 18.6
0.36 f 0.49 * 0.61= 0.41 * 0.58 + 0~53 f
IRb
346nm
11.13
Tryptic hydrolysate Fragment a-c Domain d Fragment ef Domain g Domain h 4 Standard deviation (rz = 5).
0.02 0.05 0.01 0.03 0.04 0.08
4.86 3.75 3.99 3.53 4.19 4-43
Constant Gielens et aL
borate
b
C
I
900
850
I
I
250
300
Avtcm-'I and the v(Cu-N) band at 270 cm-1 of (a) domaing, 21.4 mghl, 0.74 mbf Cu, (IJ) domain h, 18.6 m&l, 0.61 m&i Cu. and (c) tenth molecules of ~c-haemocyanin, 19.0 mghnl, 0.69 mM Cu, using the 457.9 nm line of an argon ion laser. The slit width is 6 cm-1 and the scamdug speed is 10 an-1 FIGURE
3. Resonance Raman qxc?mm
showing the borate reference peak at 873 cm-l
Inin=?. c&s of &&aemocyanin (Figure 3). The important lowering of the intensity of this band suggests a m$ifkaticn of the orientation of the ligandsof copper, likely imidazole groups, which probably becomes more asymmetric.
CONCLUSIONS The results of this study co&m that the tryptic fragments contain well-preserved active sites, as shown by the preservation of the ~$0-0) v&ration. The orientation of the ligands of Cu may somewhat differ in the fragments and in the tenth molecules of &-haemocyanin, as suggested by the decrease of the intensity of the v(C!u-N) vibration, tie are gnGq2 to professor D- ShCver, Evanston, IL. for he&@ debted to the N&naal Fends voor Wetemdtappelijk Ondenoek
disacszions. The authors are infor a f&w&p of Qangetteki
Raman Resonance of Haemocyanin
Fragments
47
Navorsw” [GM), to the Bel,#an Government [Convention No. 76.81l.II.4). and to the Fonds voor CNIectief Fundomenteel Ondersoek. Brussels, Belgium. for research grants (Contract No. 2.0016.76).
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Received December I, 1979; revised January 2,198O