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Preparation of an affinity resin for odorants by coupling odorant binding protein from bovine nasal mucosa to Sepharose 4B
Journal of Biotechnology, 30 (1993) 225-230
225
© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00
BIOTEC 00888
Preparation of an affinity resin for odorants by coupling odorant binding protein from bovine nasal mucosa to Sepharose 4B L. Bussolati, R. R a m o n i , S. Grolli, G. D o n o f r i o a n d E. Bignetti Institute of Veterinary Biochemistry, University of Parma, Italy
(Received 10 October 1992;revision accepted 16 November 1992)
Summary The soluble Odorant Binding protein (OBP) of bovine nasal mucosa is a dimer of non-covalently bound subunits that specifically recognizes several odorants of different chemical classes. Monomerization and binding activity impairment are concomitant reversible processes that occur below pH 6.0. In order to avoid this inconvenience, a mild pH condition has been chosen to couple native OBP to CNBr-activated Sepharose 4B resin. Above pH 6.0, the OBP-Sepharose matrix shows high specificity and affinity for odorants, thus opening new perspectives in odorant-sensor technology. Odorant-Binding protein; Odorant; Affinity chromatography
Introduction A major concern of food industry is to adopt a 'total quality' strategy, an approach that includes also the evaluation of organoleptic properties. Olfactory and gustatory stimuli are now considered to act not only as flavours for a better palatability, but also as indicators of correct food processing and storage. In this wide area, food science and technology are moving towards the logic of an 'objective' evaluation of odours that might be rapid, reliable and possibly Correspondence to." E. Bignetti, Ist. BiochimicaVeterinaria, Universit~ degli Studi, v. del Taglio 8, 43100
Parma, Italy.
226
on-line. In a critical review, Pelosi (1989) summarized the actual methods for the analysis of such compounds and grouped them mainly into either psychophysical or instrumental modalities, but no examples of odorant biosensors seemed to be available so far. Strictly speaking, even the new commercially available Fragrance Sensor SF-105 (Toyo Corporation, Japan) which is based on a synthetic lipid bilayer coupled to a piezoelectric device, cannot be considered an odorant biosensor. Recent studies we have carried out on molecular aspects of vertebrate olfaction, led us to the purification and characterization of an Odorant Binding protein (OBP), secreted with mucus by glands in cow nasal mucosa (see for a review: Bignetti et al., 1988). Taking advantage of this, we developed a novel OBP-based affinity matrix for odorants that might be used as a tool in odorant-biosensor technology. Materials and Methods
Materials Tritium labelled bell pepper odorant 2-isobutyl-3-methoxy pyrazine (Pyrazine*; 1700 cpm pmol-l) and geosmin (earthy odorant) were gifts of Dr P. Pelosi (Istituto di Industrie Agrarie, Pisa, Italy). Unlabelled (cold) pyrazine, (-)-carvone (minthy odorant) and thymol (disinfectant odorant) were purchased by Aldrich Chemie (Germany). CNBr-activated Sepharose 4B resin, /3-1actoglobulin, ovalbumin and soybean trypsin inhibitor were from Sigma Chemical Co. (USA). Chromopak column for gel permeation was from Baker Inc. (USA). Fast Protein Liquid Chromatography (FPLC) was from Pharmacia (Sweden)./3-Counter was a Tri-Carb 300 from Packard Instruments Co. (USA). Other chemicals were of the best commercial grades.
Odorant Binding protein Bovine Odorant Binding protein (OBP) with the expected pyrazine binding activity (1 pyrazine bound/OBP dimer; K d = 3 /zM) was purified from cow nasal mucosa according to our method (Bignetti et al., 1985). The protein (10-15 mg ml- 1) was microcrystallized at 4°C in a solution of Tris-HCl buffer 10 mM pH 7.4, ethanol 10% (v/v), and stored at 4°C.
Effect of pH on OBP structure and function Samples of OBP (5 gM) were dialysed overnight at 4°C against phosphate buffer 50 mM and NaCI 200 mM, adjusted to pH ranging from 3 to 11 by HCI or NaOH additions. At different pH, binding activity was tested in the presence of half-saturating pyrazine (3 /zM) by our method (Bignetti et al., 1985). Dimer/ monomer equilibrium in OBP was evaluated by gel permeation chromatography on a Chromopack column in FPLC (each run was less than 50 min). At each pH, the column was calibrated with ovalbumin and soybean trypsin inhibitor (respectively of 45 and 21.5 kDa). In control experiments, eluted OBP peaks were rechromatographed in order to demonstrate that gel permeation did not perturb the dimer/monomer equilibrium assessed overnight.
227
Affinity column for odorants Pure OBP was coupled to CNBr-Sepharose resin with a crucial modification of our procedure (Bignetti et al., 1987). A typical preparation required 20 mg of OBP in 3 ml of coupling buffer (NaHCO 3 0.2 M pH 9.0), to which 300 mg of dry resin were added. After gentle stirring for 24 h at room temperature, the resin was packed in a 1 ml column and washed with 30 ml of coupling buffer: in this step, 9 mg of protein was released. The column was not washed with acidic buffer but it was let to stand in Tris-HCl 10 mM pH 7.4 (working buffer) at 4°C overnight. In this step, no further release of OBP was observed. Affinity chromatography in working buffer was carried out in three compulsory steps: (1) loading a mixture (150 /xl-1 ml) of tritium-labelled pyrazine (3 x 10-1°-3 x 10 -8 M) and cold odorant (0-3 x 10 -6 M); (2) washing the column (10 times the volume of the column); (3) adding (150 ~1-1 ml) cold odorant (10 -3 M).
Results and Discussion
Odorant Binding protein from cow nasal mucosa is a dimer of non-covalently associated subunits that binds only one pyrazine molecule with a dissociation constant K a = 3/zM (Bignetti et al., 1985). The existence of a central binding site created by both subunits was already anticipated on theoretical bases (Bignetti et al., 1988), and some X-ray experiments now in progress on OBP crystals seem to sustain that view. Therefore, we postulated that the weakening of OBP monomer/ monomer interactions due to external reasons should interfere with its function. In Fig. 1, the results indicate that OBP exists in a dimer/monomer equilibrium that can be selectively perturbed by pH. In particular, the acidic pK of dimer dissociation, in our experimental conditions, corresponds to pH 4.9. Moreover, impairment of pyrazine binding activity reversibly follows almost the same pH dependence of subunit dissociation, with an apparent pK corresponding to pH 4.5. The results, together, suggest that the dimer and not the monomer is competent for the function. Taking advantage of these studies, we found out correct experimental conditions in order to immobilize active OBP to CNBr-Sepharose 4B. As recommended by other protocols (Bignetti et al., 1987), the immobilization reaction was carried out with large excess of OBP with respect to resin equivalents but extensive washings with buffers below pH 6.0 after protein linkage, were omitted. The OBP coupling efficiency was 30 mg g-1 of dry resin, that corresponds to about 300/~M OBP in swollen state. Active OBP-Sepharose resin was packed into columns of variable size (150/zl-1 ml) and tested for odorant affinity. Negative controls were run with OBP-resins that lost about half of the protein upon acidic treatment and with resins containing covalently attached fl-lactoglobulin, a protein structurally homologous to OBP (Tirindelli et al., 1989). An example of odorant binding assay is reported in Fig. 2. A 150/~1 solution of cold pyrazine (up to 3/zM) pre-mixed with radiolabelled pyrazine (10 -8 M) was
228 [monomerl/[OBPol
specific activity
100 i
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pit Fig. 1. Bovine OBP structure and function dependence on pH. (A) Dimer/monomer equilibrium of OBP at different pH evaluated by fast gel-permeation chromatography. (B) Specific binding activity of OBP towards pyrazine odorant in function of pH. Data points have been interpolated by means of a best-fitting program for pK a determination (dotted lines).
l o a d e d i n t o a 1 5 0 / z l c o l u m n . T r a c e s o f r a d i o a c t i v e o d o r a n t w e r e n o t e l u t e d in this step, n o r t h e y c o u l d b e r e l e a s e d by an e x t e n s i v e w a s h i n g . T h e r e t a i n e d r a d i o a c t i v e odorant could be competitively displaced and eluted only after loading 150/xl of 1
4
cpm x lO00 A B
C
°41
LoADW A S .
,
t, mt~o~
t was.
:,
4
(mi)
Fig. 2. Affinity-chromatography of odorants on a 150 p.l column of bovine OBP coupled to Sepharose 4B. At time 0, addition of 150 ~1 of a labelled/cold pyrazine mixture (3/zM, 12000 cpm). After a 1.7 ml wash, specific elution of the retained radiolabelled odorant by adding 600 /~1 of: (A) 1 mM cold pyrazine; (B) 1 mM thymol; (C) 1 mM (-)-carvone. Geosmin was uneffective.
229 mM cold pyrazine (150 nmol) which could saturate OBP (40 nmol). Profiting of the high stability and reproducibility of the resin, competitive displacement experiments could be successfully repeated on the same column by substituting carvone and thymol (1 mM) for cold pyrazine. Specificity of the column was also tested with the earthy odorant geosmin. Among OBP ligands, synthetic geosmin was initially considered the strongest pyrazine competitor ( g i = 0.02 /.tM) by some authors (Pelosi and Tirindelli, 1989; Cavaggioni et al., 1990). Moreover, due to the presence of suspicious solvent in that preparation, efforts were made to obtain extremely pure earthy odorants (Finato et al., 1992). In our hands, a new batch of geosmin did not displace pyrazine from OBP either in solution or in the affinity column. Finally, we tentatively evaluated pyrazine detection threshold of our affinity resin. The OBP concentration in the column is about two orders of magnitude higher than the dissociation constant of pyrazine binding in solution. Assuming that no changes in intrinsic affinity had occurred during the immobilization step, detection threshold of our column should be very low. Due to restriction mainly posed by the low specific activity of the radiolabelled pyrazine, we could not handle less than 4.5 X 10 -14 mol of it in a volume of 150/zl (3 × 10 -1° M). More than 90% of the radioactivity of this sample could be specifically retained, even by the smallest available column (150 /zl). If one considers that about 2.4 × 10 -13 moles of pyrazine in approx. 20 ml of water (1.2 x 10-11 M) are required for the psycophysical recognition in so called human "sniff-test" (Amoore and Buttery, 1978; Buttery et al., 1969), one should conclude that our resin is extremely efficient at least in odorant binding. We are now trying to couple this affinity matrix to a device that might recognize the ligand and transduce it in a non-radioactive measurable signal.
Acknowledgements We are indebted to A. Cavaggioni for helpful discussion and cooperation and to P. Pelosi for giving us geosmin and tritium-labelled pyrazine. Research supported by National Research Council of Italy, special project RAISA, sub-project 4, paper 650.
References Amoore, J.E. and Buttery, R.G. (1978) Partition coefficientsand comparative olfactometry. Chem. Sens. Flavour3, 57-71. Bignetti, E., Cavaggioni,A., Pelosi, P., Persaud, K.C., Sorbi, R. and Tirindelli, R. (1985) Purification and characterization of an odorant-bindingprotein from cow nasal mucosa. Eur. J. Biochem. 149, 227-231.
230 Bignetti, E., Damiani, G., De Negri, P., Ramoni, R., Avanzini, F., Ferrari, G. and Rossi, G.L. (1987) Specificity of an immunoaffinity column for odorant-binding protein from cow nasal mucosa. Chem. Senses 12, 601-608. Bignetti, E., Cattaneo, P., Cavaggioni, A., Damiani, G. and Tirindelli, R. (1988) The pyrazine-binding protein and olfaction. Comp. Biochem. Physiol. 90B, 1-5. Buttery, R.G., Seifect, R.M., Ludin, R.E., Guadagni, D.G. and Ling, L.C. (1969) Characterization of an important aroma component of bell peppers. J. Agric. Food Chem. 17, 1322-1327. Cavaggioni, A., Findlay, J.B.C. and Tirindelli, R. (1990) Ligand bindig characteristics of homologous rat and mouse urinary proteins and pyrazine binding proteins from calf. Comp. Biochem. Physiol. 96B, 513-520. Finato, B., Lorenzi, R. and Pelosi, P. (1992) Synthesis of new earthy odorants. J. Agric. Food Chem. 40, 857-859. Pelosi, P. (1989) Towards an objective evaluation of odours in food. Ital. J. Food Sci. 1, 5-22. Peiosi, P. and Tirindelli, R. (1989) Structure/activity studies and characterization of an odorant binding protein. In: Brand et al. (Eds.) Chemical Senses, 1. Marcel Dekker, New York, pp. 207-226. Tirindelli, R., Keen, J.N., Cavaggioni, A., Eliopoulos, E.E. and Findlay, J.B.C. (1989) Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa. Eur. J. Biochem. 185, 569-572.
225
© 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00
BIOTEC 00888
Preparation of an affinity resin for odorants by coupling odorant binding protein from bovine nasal mucosa to Sepharose 4B L. Bussolati, R. R a m o n i , S. Grolli, G. D o n o f r i o a n d E. Bignetti Institute of Veterinary Biochemistry, University of Parma, Italy
(Received 10 October 1992;revision accepted 16 November 1992)
Summary The soluble Odorant Binding protein (OBP) of bovine nasal mucosa is a dimer of non-covalently bound subunits that specifically recognizes several odorants of different chemical classes. Monomerization and binding activity impairment are concomitant reversible processes that occur below pH 6.0. In order to avoid this inconvenience, a mild pH condition has been chosen to couple native OBP to CNBr-activated Sepharose 4B resin. Above pH 6.0, the OBP-Sepharose matrix shows high specificity and affinity for odorants, thus opening new perspectives in odorant-sensor technology. Odorant-Binding protein; Odorant; Affinity chromatography
Introduction A major concern of food industry is to adopt a 'total quality' strategy, an approach that includes also the evaluation of organoleptic properties. Olfactory and gustatory stimuli are now considered to act not only as flavours for a better palatability, but also as indicators of correct food processing and storage. In this wide area, food science and technology are moving towards the logic of an 'objective' evaluation of odours that might be rapid, reliable and possibly Correspondence to." E. Bignetti, Ist. BiochimicaVeterinaria, Universit~ degli Studi, v. del Taglio 8, 43100
Parma, Italy.
226
on-line. In a critical review, Pelosi (1989) summarized the actual methods for the analysis of such compounds and grouped them mainly into either psychophysical or instrumental modalities, but no examples of odorant biosensors seemed to be available so far. Strictly speaking, even the new commercially available Fragrance Sensor SF-105 (Toyo Corporation, Japan) which is based on a synthetic lipid bilayer coupled to a piezoelectric device, cannot be considered an odorant biosensor. Recent studies we have carried out on molecular aspects of vertebrate olfaction, led us to the purification and characterization of an Odorant Binding protein (OBP), secreted with mucus by glands in cow nasal mucosa (see for a review: Bignetti et al., 1988). Taking advantage of this, we developed a novel OBP-based affinity matrix for odorants that might be used as a tool in odorant-biosensor technology. Materials and Methods
Materials Tritium labelled bell pepper odorant 2-isobutyl-3-methoxy pyrazine (Pyrazine*; 1700 cpm pmol-l) and geosmin (earthy odorant) were gifts of Dr P. Pelosi (Istituto di Industrie Agrarie, Pisa, Italy). Unlabelled (cold) pyrazine, (-)-carvone (minthy odorant) and thymol (disinfectant odorant) were purchased by Aldrich Chemie (Germany). CNBr-activated Sepharose 4B resin, /3-1actoglobulin, ovalbumin and soybean trypsin inhibitor were from Sigma Chemical Co. (USA). Chromopak column for gel permeation was from Baker Inc. (USA). Fast Protein Liquid Chromatography (FPLC) was from Pharmacia (Sweden)./3-Counter was a Tri-Carb 300 from Packard Instruments Co. (USA). Other chemicals were of the best commercial grades.
Odorant Binding protein Bovine Odorant Binding protein (OBP) with the expected pyrazine binding activity (1 pyrazine bound/OBP dimer; K d = 3 /zM) was purified from cow nasal mucosa according to our method (Bignetti et al., 1985). The protein (10-15 mg ml- 1) was microcrystallized at 4°C in a solution of Tris-HCl buffer 10 mM pH 7.4, ethanol 10% (v/v), and stored at 4°C.
Effect of pH on OBP structure and function Samples of OBP (5 gM) were dialysed overnight at 4°C against phosphate buffer 50 mM and NaCI 200 mM, adjusted to pH ranging from 3 to 11 by HCI or NaOH additions. At different pH, binding activity was tested in the presence of half-saturating pyrazine (3 /zM) by our method (Bignetti et al., 1985). Dimer/ monomer equilibrium in OBP was evaluated by gel permeation chromatography on a Chromopack column in FPLC (each run was less than 50 min). At each pH, the column was calibrated with ovalbumin and soybean trypsin inhibitor (respectively of 45 and 21.5 kDa). In control experiments, eluted OBP peaks were rechromatographed in order to demonstrate that gel permeation did not perturb the dimer/monomer equilibrium assessed overnight.
227
Affinity column for odorants Pure OBP was coupled to CNBr-Sepharose resin with a crucial modification of our procedure (Bignetti et al., 1987). A typical preparation required 20 mg of OBP in 3 ml of coupling buffer (NaHCO 3 0.2 M pH 9.0), to which 300 mg of dry resin were added. After gentle stirring for 24 h at room temperature, the resin was packed in a 1 ml column and washed with 30 ml of coupling buffer: in this step, 9 mg of protein was released. The column was not washed with acidic buffer but it was let to stand in Tris-HCl 10 mM pH 7.4 (working buffer) at 4°C overnight. In this step, no further release of OBP was observed. Affinity chromatography in working buffer was carried out in three compulsory steps: (1) loading a mixture (150 /xl-1 ml) of tritium-labelled pyrazine (3 x 10-1°-3 x 10 -8 M) and cold odorant (0-3 x 10 -6 M); (2) washing the column (10 times the volume of the column); (3) adding (150 ~1-1 ml) cold odorant (10 -3 M).
Results and Discussion
Odorant Binding protein from cow nasal mucosa is a dimer of non-covalently associated subunits that binds only one pyrazine molecule with a dissociation constant K a = 3/zM (Bignetti et al., 1985). The existence of a central binding site created by both subunits was already anticipated on theoretical bases (Bignetti et al., 1988), and some X-ray experiments now in progress on OBP crystals seem to sustain that view. Therefore, we postulated that the weakening of OBP monomer/ monomer interactions due to external reasons should interfere with its function. In Fig. 1, the results indicate that OBP exists in a dimer/monomer equilibrium that can be selectively perturbed by pH. In particular, the acidic pK of dimer dissociation, in our experimental conditions, corresponds to pH 4.9. Moreover, impairment of pyrazine binding activity reversibly follows almost the same pH dependence of subunit dissociation, with an apparent pK corresponding to pH 4.5. The results, together, suggest that the dimer and not the monomer is competent for the function. Taking advantage of these studies, we found out correct experimental conditions in order to immobilize active OBP to CNBr-Sepharose 4B. As recommended by other protocols (Bignetti et al., 1987), the immobilization reaction was carried out with large excess of OBP with respect to resin equivalents but extensive washings with buffers below pH 6.0 after protein linkage, were omitted. The OBP coupling efficiency was 30 mg g-1 of dry resin, that corresponds to about 300/~M OBP in swollen state. Active OBP-Sepharose resin was packed into columns of variable size (150/zl-1 ml) and tested for odorant affinity. Negative controls were run with OBP-resins that lost about half of the protein upon acidic treatment and with resins containing covalently attached fl-lactoglobulin, a protein structurally homologous to OBP (Tirindelli et al., 1989). An example of odorant binding assay is reported in Fig. 2. A 150/~1 solution of cold pyrazine (up to 3/zM) pre-mixed with radiolabelled pyrazine (10 -8 M) was
228 [monomerl/[OBPol
specific activity
100 i
a
A 75
"
o
g
m i
a
I
a
m
n
m
I
II~xm
a
•
I I I
B
I
m
a
i
A
Z3,"
75
~.
%
50
25
25 ~'.
& •
3
A %
I
I
4
5
i a~|
l
6
7
8
9
10
11
pit Fig. 1. Bovine OBP structure and function dependence on pH. (A) Dimer/monomer equilibrium of OBP at different pH evaluated by fast gel-permeation chromatography. (B) Specific binding activity of OBP towards pyrazine odorant in function of pH. Data points have been interpolated by means of a best-fitting program for pK a determination (dotted lines).
l o a d e d i n t o a 1 5 0 / z l c o l u m n . T r a c e s o f r a d i o a c t i v e o d o r a n t w e r e n o t e l u t e d in this step, n o r t h e y c o u l d b e r e l e a s e d by an e x t e n s i v e w a s h i n g . T h e r e t a i n e d r a d i o a c t i v e odorant could be competitively displaced and eluted only after loading 150/xl of 1
4
cpm x lO00 A B
C
°41
LoADW A S .
,
t, mt~o~
t was.
:,
4
(mi)
Fig. 2. Affinity-chromatography of odorants on a 150 p.l column of bovine OBP coupled to Sepharose 4B. At time 0, addition of 150 ~1 of a labelled/cold pyrazine mixture (3/zM, 12000 cpm). After a 1.7 ml wash, specific elution of the retained radiolabelled odorant by adding 600 /~1 of: (A) 1 mM cold pyrazine; (B) 1 mM thymol; (C) 1 mM (-)-carvone. Geosmin was uneffective.
229 mM cold pyrazine (150 nmol) which could saturate OBP (40 nmol). Profiting of the high stability and reproducibility of the resin, competitive displacement experiments could be successfully repeated on the same column by substituting carvone and thymol (1 mM) for cold pyrazine. Specificity of the column was also tested with the earthy odorant geosmin. Among OBP ligands, synthetic geosmin was initially considered the strongest pyrazine competitor ( g i = 0.02 /.tM) by some authors (Pelosi and Tirindelli, 1989; Cavaggioni et al., 1990). Moreover, due to the presence of suspicious solvent in that preparation, efforts were made to obtain extremely pure earthy odorants (Finato et al., 1992). In our hands, a new batch of geosmin did not displace pyrazine from OBP either in solution or in the affinity column. Finally, we tentatively evaluated pyrazine detection threshold of our affinity resin. The OBP concentration in the column is about two orders of magnitude higher than the dissociation constant of pyrazine binding in solution. Assuming that no changes in intrinsic affinity had occurred during the immobilization step, detection threshold of our column should be very low. Due to restriction mainly posed by the low specific activity of the radiolabelled pyrazine, we could not handle less than 4.5 X 10 -14 mol of it in a volume of 150/zl (3 × 10 -1° M). More than 90% of the radioactivity of this sample could be specifically retained, even by the smallest available column (150 /zl). If one considers that about 2.4 × 10 -13 moles of pyrazine in approx. 20 ml of water (1.2 x 10-11 M) are required for the psycophysical recognition in so called human "sniff-test" (Amoore and Buttery, 1978; Buttery et al., 1969), one should conclude that our resin is extremely efficient at least in odorant binding. We are now trying to couple this affinity matrix to a device that might recognize the ligand and transduce it in a non-radioactive measurable signal.
Acknowledgements We are indebted to A. Cavaggioni for helpful discussion and cooperation and to P. Pelosi for giving us geosmin and tritium-labelled pyrazine. Research supported by National Research Council of Italy, special project RAISA, sub-project 4, paper 650.
References Amoore, J.E. and Buttery, R.G. (1978) Partition coefficientsand comparative olfactometry. Chem. Sens. Flavour3, 57-71. Bignetti, E., Cavaggioni,A., Pelosi, P., Persaud, K.C., Sorbi, R. and Tirindelli, R. (1985) Purification and characterization of an odorant-bindingprotein from cow nasal mucosa. Eur. J. Biochem. 149, 227-231.
230 Bignetti, E., Damiani, G., De Negri, P., Ramoni, R., Avanzini, F., Ferrari, G. and Rossi, G.L. (1987) Specificity of an immunoaffinity column for odorant-binding protein from cow nasal mucosa. Chem. Senses 12, 601-608. Bignetti, E., Cattaneo, P., Cavaggioni, A., Damiani, G. and Tirindelli, R. (1988) The pyrazine-binding protein and olfaction. Comp. Biochem. Physiol. 90B, 1-5. Buttery, R.G., Seifect, R.M., Ludin, R.E., Guadagni, D.G. and Ling, L.C. (1969) Characterization of an important aroma component of bell peppers. J. Agric. Food Chem. 17, 1322-1327. Cavaggioni, A., Findlay, J.B.C. and Tirindelli, R. (1990) Ligand bindig characteristics of homologous rat and mouse urinary proteins and pyrazine binding proteins from calf. Comp. Biochem. Physiol. 96B, 513-520. Finato, B., Lorenzi, R. and Pelosi, P. (1992) Synthesis of new earthy odorants. J. Agric. Food Chem. 40, 857-859. Pelosi, P. (1989) Towards an objective evaluation of odours in food. Ital. J. Food Sci. 1, 5-22. Peiosi, P. and Tirindelli, R. (1989) Structure/activity studies and characterization of an odorant binding protein. In: Brand et al. (Eds.) Chemical Senses, 1. Marcel Dekker, New York, pp. 207-226. Tirindelli, R., Keen, J.N., Cavaggioni, A., Eliopoulos, E.E. and Findlay, J.B.C. (1989) Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa. Eur. J. Biochem. 185, 569-572.