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Quantification of muscle proteolytic enzymes by immunochemical techniques
M. A. Sentandreu1, O. Chantreau1, L. Aubry1, D. Levieux2 and A. Ouali1
1 – INTRODUCTION
Inconsistency and unpredictable variability in the final quality of meat are among the main problems that nowadays meat industry has to solve in order to satisfy consumer demands in terms of product safety, texture and flavour. These problems come undoubtedly from the biological complexity of skeletal muscle.
Endogenous muscle proteolytic enzymes are assumed to play a basic role in this context question, since they are responsible of the post-mortem myofibrillar disruption, which is a change directly related with the development of meat texture and flavour. So, the quantification of some proteolytic enzymes in muscle has been proposed as a way to explain this variability and this has been traditionally done by measurement of their enzymatic activities in muscle crude extracts.
However these measurements are imprecise and complicated because of the absence of specific substrates for individual enzymes. An absolute prerequisite for their quantification by activity measurement is a previous fractionation of the tissue extract, a procedure taking a long time and limiting extensively the number of sam- ples that can be analysed in parallel. We think of importance to develop alternative methods enabling more accurate and specific quantification of muscle proteolytic enzymes and avoiding the fractionation step. Such goal could be achieved by using immunochemical techniques as those described in the present work.
2 – IMMUNOCHEMICAL TECHNIQUES
There are two techniques that can be especially interesting for Meat Scien- tists. The first one referred to as ELISA (Enzyme Linked Immunosorbent Assay) is already currently used for medical diagnostic purposes. The second one is known as Radial Immunodiffusion or Mancini technique (MANCINI et al., 1965).
1. Muscle Biochemistry Group, SRV, INRA-Theix, 63122 Saint Genès Champanelle, France.
2. Immuno Chemistry Group, SRV, INRA-Theix, 63122 Saint Genès Champanelle, France.
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ELISA
These systems utilise a solid support, as a microtitre plate, in which the anti- body or the antigen is absorbed onto the support and so only the components reacting specifically with the absorbed substance are retained in the well. Then, absorbed substances are specifically quantified by the use of different detec- tion systems. The different modalities of the Enzyme Linked Immunosorbent Assay (ELISA) are probably the most interesting for use in Meat Science because they are very sensitive and do not required expensive equipment.
Two modalities are particularly interesting for our purpose:
– Indirect ELISA: The crude extract is coated directly into the plate wells.
The target compound is then recognised by a specific antibody (primary antibody), which is itself recognised by a second antibody (anti-immuno- globulin antibody) covalently coupled to an enzyme giving a coloured reac- tion. Colour intensity at a determined wavelength is directly proportional to the antigen concentration.
The method is relatively easy to use and does not take a long time. Howe- ver, the high complexity of muscle homogenates makes it difficult to set it up for biological markers because a lot of interference can occur during antigen absorption or with the primary antibody. In such cases an initial extraction/concentration of the antigen from crude extract is required prior to develop the immunoassay.
– Sandwich ELISA: In this modality, a highly specific antibody against the substance of interest (antigen) is firstly absorbed into the wells of the plate.
Antigen is then captured from the crude extract, which remains absorbed to the plate, whereas all the rest is eliminated by washing. A second, peroxydase-labelled, polyclonal antibody directed to the antigen is now required to be further revealed by the peroxidase action on a substrate giving a coloured reaction. Sandwich ELISA provides a very high sensiti- vity and specificity, allowing to work directly with complex matrix as mus- cle crude extracts and being able to detect antigens in the nanomolar range.
Radial immunodiffusion
This technique is based on the precipitation of the antigen-antibody complex on a 1-2% gelose gel. Antibody against the target compound is solubilised in the gel at a determined concentration, whereas muscle crude extract is deposed in a well into the gel. The target compound (antigen) contained in the muscle extract diffuses into the gel, forming a precipitating arch at the point of equivalence. Area contained in the precipitating ring is then proportional to the concentration of the antigen in the crude extract.
This technique has the advantage to be easy to use and requires neither expensive equipment nor trained staff, allowing the processing of a great num- ber of samples. However, the sensitivity of this method is limited since the anti- gen to be quantified must be present in the crude extract at a concentration higher than 1-10 µg/ml, depending on the molecular mass of the antigen.
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3 – SOME EXAMPLES OF IMMUNOCHEMICAL APPLICATIONS TO THE QUANTIFICATION OF PEPTIDASES
3.1 Sandwich ELISA for the quantification of bovine cathepsin L
Cathepsin L is the most powerful of the lysosomal peptidases (BARRETT and KIRSCHKE, 1981), being one of the potential contributors to postmor- tem protein degradation. Determination of cathepsin L levels in muscle could be an interesting biological marker of meat texture. However, its quantification by determining its enzyme activity in crude extracts actually is not possible since there is no specific substrate for this endopeptidase. The concentration in muscle homogenates is also very low, in the nanomolar range, so an ELISA assay could be a way to solve the problem and correlate cathepsin L levels with meat texture.
To achieve this goal, a polyclonal antibody against highly purified bovine cathepsin L has been raised in rabbits. From the total antiserum, the IgG fraction was purified by chromatogaphy on a DEAE-Sepharose column. Part of the IgG fraction was biotinylated and used as the secon- dary antibody. This was further revealed using peroxydase-labeled avidin.
The quality of this antibody in terms of affinity, specificity and cross-reacti- vity has been certified by western-blot (data not shown). The standard curve obtained with various concentration of purified cathepsin L ranging from 0 to 10 µg/ml is presented in Figure 1. The primary antibody coated in the wells and used at a dilution of 1/1000 is the IgG fraction and the secondary antibody is the biotinylated IgG fraction. As can be seen, at a concentration as low as 10 ng/ml we can already appreciate a positive res- ponse with the present sandwich ELISA assay. So, the concentration of cathepsin L which can be accurately determined with this assay is in the range of 10 to 100 ng/ml. In the near future we will apply this test to deter- mine the cathepsin L concentration in crude extracts of different types of bovine muscles and in muscles of different animals, in order to establish a correlation with meat texture.
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Figure 1
Quantification of bovine cathepsin L by using a polyclonal antibody raised against the purified enzyme in a sandwich ELISA.
Cathepsin L concentration is expressed in logarithm scale.
3.2 Quantification of the 20S proteasome by radial immunodiffusion The 20S proteasome is a 700 kDa multi-enzymatic complex present in ske- letal muscle in a relatively high concentration, ranging from 200 to 350 µg per gram of wet muscle (DUTAUD, D., 1998). Some recent investigations have pointed out a major role of the 20S proteasome in postmortem tenderisation of high pH meat and slow-twitch muscles, indicating its high hydrolytic potential in postmortem muscle (OUALI, 1999). Its complex structure explains the exis- tence of three different enzymatic activities, making impossible the quantifica- tion of the 20S proteasome in muscle by measurement of the enzymatic activities. Due to its important concentration in muscle, an immunoprecipitation test for the quantification of 20S proteasome seemed to be an adequate, simple and rapid way to achieve this goal. Additionally, an ELISA test, with an enhan- ced sensitivity, has been also developed for the quantification of proteasome in both bovine tissues and human serum (DUTAUD et al., 2002).
– Quantification of 20S proteasome by radial immunodiffusion: A polyclonal antibody against the whole purified proteasome from bovine muscle was raised in rabbits. As for cathepsis L, the quality of the antiserum was assessed by immunoelectrophoresis and western-blot (DUTAUD, D., 1998). This polyclonal antibody is then homogenised into the gelose (20- 30 µl serum/5ml gelose). Into the holes (3µl), different concentrations of purified proteasome together with muscle extracts are deposed, leaving the diffusion through the gelose during 24 hours (figure 2A). The diameter of each precipitating ring is obtained at the equivalence point between proteasome and antibody concentrations. From the standard curve of pre- cipitating rings corresponding to different concentrations ranging from 25
0 1 10 100 1 000 10 000 100 000
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 Cathepsin L concentration (ng/ml)
Absorbance 492 nm
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to 200 µg of pure proteasome (figure 2B), we obtained a proteasome con- centration of 250 and 200 µg per gram of muscle in Longissimus dorsi and Supraspinatus muscles, respectively.
Figure 2
Quantification of 20S proteasome by radial immunodiffusion in solidified gelose.
The surface of precipitation circles of the antibody/antigen complex is proportional to the proteasome concentration.
(A) Different concentrations of pure proteasome (antigen) and two muscle crude extracts are deposed on the gelose plate.
(B) Standard curve for quantification of 20S proteasome in both Longissimus Dorsi and Supraspinatus muscle crude extracts is obtained by plotting the precipitation
diameters against the square root of known proteasome concentrations 40
60 80 100 120 140
2 4 6 8 10 12 14 16
B
[Proteasome (µg/ml)]1/2
Precipitate diameter (mm)
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CONCLUSION
The examples presented here for cathepsin L and proteasome show the advantage that immunological techniques can afford to meat biochemistry, hel- ping us to better understand the complex mechanisms of meat tenderisation. By developing specific and sensitive enough quantification test we could advance in knowing the role of each of the enzyme groups implicated in meat tenderisation.
We would be able to work with crude extracts with relatively inexpensive equip- ment and non-specialised personnel, allowing the treatment of a large number of samples at industrial scale. Biological markers of tenderness without biological activity could also be determined by one of the exposed methods. In all cases, the availability of high quality antibodies is decisive for a good quantification.
Depending on the nature of the biological marker, we will have to select the most convenient assay in terms of simplicity, sensitivity and disposable equipment.
ACKNOWLEDGEMENTS
This work was supported by a Marie Curie Individual Fellowship attributed to Sentandreu, M. A.
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DUTAUD D., AUBRY L., HENRY L., LEVIEUX D., HENDIL K.B., KUEHN L., BUREAU J.P., Ouali A., 2002. Development and evalua- tion of a sandwich ELISA for quantification
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