- Email: [email protected]
[8] Extraction of soluble enzymes from higher plants
62
GENERAL PREPARATIVE PROCEDURES
~]
which exerts generalized action on proteins, often with denaturation. Recently, polyelectrolytes (i.e., a dimethyl aminoethyl acryllate polymer) active in low concentration have been introduced by Puck a9 for the liberation of bacteriaphage and promise also to be useful in the liberation of enzymes. These methods have not, however, been explored sufficiently to allow general application. s9T. T. Puck, Cold Spring Harbor Symposia Quant. Biol. 18, 153 (1953).
[8] Extraction of Soluble Enzymes from Higher P l a n t s By ALVIN NASON
Preparation Principle. The procedures outlined below are intended to extract soluble protein containing maximum enzymatic activity from plant cells under conditions least favorable for protein denaturation. The use of a buffered extracting solution, generally ranging on the alkaline side, is usually desirable. The addition of sulfhydryl compounds, other reducing substances, or metal-binding agents may enhance the extraction, activity, and stability of some enzymes, depending on their properties. I Highspeed centrifugation is necessary to free the extract of chloroplast fragments, grana, and mitochondria. Reagents 0.1 M potassium phosphate buffer, pH 7.5, with or without 10-3 M cysteine or Versene (ethylenediaminetetraacetic acid). Chilled acetone ( - 15°). Procedures. The variety of procedures which have been used for obtaining soluble cell-free enzyme extracts from higher plants can be classified into the following three main groups. In most cases either fresh or frozen tissue may be used as starting material, depending on the activity obtained in the final preparation. In the case of seed materials, the seed may be soaked overnight by covering with tap water and then extracting essentially by the methods below; or the dried seeds may first be ground or milled to a powder and then extracted essentially by method 2. METHOD 1. Extracts of pulpy tissues such as storage organs (e.g., the carrot or potato) can be prepared by first passing the tissue through a meat grinder. This is followed by pressing out the juice by hand or mechanical press through muslin or three to four layers of cheesecloth. An active extract of starch phosphorylase has been prepared from potato 1See M. Gibbs, Vol. I [62].
[8]
EXTRACTION OF SOLUBLE ENZYMES FROM HIGHER PLANTS
63
in this manner. ~ Overnight freezing and subsequent thawing of etiolated barley shoots followed by pressing by hand through cheesecloth yielded a sap for the study of the ascorbic acid system2 Centrifuging for 10 minutes at 20,000 X g further frees the liquid of suspended particles. METHOD 2. For the preparation of extracts from leaves and meristems the tissue is first ground with a cold mortar and pestle, then homogenized with a Ten Brock glass homogenizer in two to five times its weight of cold buffer, pressed through cheesecloth, and finally centrifuged at 20,000 X g for 10 minutes at 4°. ~ The Waring blendor may be substituted for the mortar and pestle, provided that the buffer is added with the leaves at this first stage. The use of alumina powder in the homogenization process was highly effective in solubilizing nitrate reductase from soybean leaves. ~ In the latter procedure the extract is made by grinding one weight of fresh leaves, three weights of cold 0.1 M K2HPO~ buffer (pH 9.0), and two weights of alumina powder (Alcoa A-301) in a Waring blendor for 2 minutes at 4 °. The mixture is further ground for 3 minutes in a Ten Brock homogenizer at 0 to 4°, then centrifuged in a Servall centrifuge at 20,000 X g for 10 minutes at 4°, yielding the clear supernatant enzyme solution. The use of sand or ground glass with the mortar and pestle excludes the subsequent use of the glass homogenizer. Such a procedure is desirable for preparing extracts from fibrous roots, since the latter tends to crack the glass homogenizer. METHOD 3. Acetone-dried powders are prepared by placing the tissue, usually leaves, in a Waring blendor, covering with at least 5 vol. of chilled acetone, and blending vigorously for 30 seconds to 1 minute. The resulting slurry is filtered through a Btichner funnel, washed with an excess of chilled acetone, and the residue spread out on filter paper and allowed to dry at room temperature. The acetone powder, which is stored in a desiccator at 4 °, may be extracted with approximately 5 to 10 vol. of phosphate buffer essentially as described in method 2. Acetone powders have the advantage of being chlorophyll-free and tend to have a minimum of gum- and resin-like material which might otherwise interfere in subsequent fractionation of the extract. R. M. McCready and W. Z. Hassid, J. Am. Chem. Soc. 66, 560 (1944). a W. O. James and J. M. Cragg, New Phytologist 42, 28 (1943). 4 A. Nason, H. A. Oldewurtel, and L. M. Propst, Arch. Biochem. and Biophys. 38, 1 (1952). H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953).
GENERAL PREPARATIVE PROCEDURES
~]
which exerts generalized action on proteins, often with denaturation. Recently, polyelectrolytes (i.e., a dimethyl aminoethyl acryllate polymer) active in low concentration have been introduced by Puck a9 for the liberation of bacteriaphage and promise also to be useful in the liberation of enzymes. These methods have not, however, been explored sufficiently to allow general application. s9T. T. Puck, Cold Spring Harbor Symposia Quant. Biol. 18, 153 (1953).
[8] Extraction of Soluble Enzymes from Higher P l a n t s By ALVIN NASON
Preparation Principle. The procedures outlined below are intended to extract soluble protein containing maximum enzymatic activity from plant cells under conditions least favorable for protein denaturation. The use of a buffered extracting solution, generally ranging on the alkaline side, is usually desirable. The addition of sulfhydryl compounds, other reducing substances, or metal-binding agents may enhance the extraction, activity, and stability of some enzymes, depending on their properties. I Highspeed centrifugation is necessary to free the extract of chloroplast fragments, grana, and mitochondria. Reagents 0.1 M potassium phosphate buffer, pH 7.5, with or without 10-3 M cysteine or Versene (ethylenediaminetetraacetic acid). Chilled acetone ( - 15°). Procedures. The variety of procedures which have been used for obtaining soluble cell-free enzyme extracts from higher plants can be classified into the following three main groups. In most cases either fresh or frozen tissue may be used as starting material, depending on the activity obtained in the final preparation. In the case of seed materials, the seed may be soaked overnight by covering with tap water and then extracting essentially by the methods below; or the dried seeds may first be ground or milled to a powder and then extracted essentially by method 2. METHOD 1. Extracts of pulpy tissues such as storage organs (e.g., the carrot or potato) can be prepared by first passing the tissue through a meat grinder. This is followed by pressing out the juice by hand or mechanical press through muslin or three to four layers of cheesecloth. An active extract of starch phosphorylase has been prepared from potato 1See M. Gibbs, Vol. I [62].
[8]
EXTRACTION OF SOLUBLE ENZYMES FROM HIGHER PLANTS
63
in this manner. ~ Overnight freezing and subsequent thawing of etiolated barley shoots followed by pressing by hand through cheesecloth yielded a sap for the study of the ascorbic acid system2 Centrifuging for 10 minutes at 20,000 X g further frees the liquid of suspended particles. METHOD 2. For the preparation of extracts from leaves and meristems the tissue is first ground with a cold mortar and pestle, then homogenized with a Ten Brock glass homogenizer in two to five times its weight of cold buffer, pressed through cheesecloth, and finally centrifuged at 20,000 X g for 10 minutes at 4°. ~ The Waring blendor may be substituted for the mortar and pestle, provided that the buffer is added with the leaves at this first stage. The use of alumina powder in the homogenization process was highly effective in solubilizing nitrate reductase from soybean leaves. ~ In the latter procedure the extract is made by grinding one weight of fresh leaves, three weights of cold 0.1 M K2HPO~ buffer (pH 9.0), and two weights of alumina powder (Alcoa A-301) in a Waring blendor for 2 minutes at 4 °. The mixture is further ground for 3 minutes in a Ten Brock homogenizer at 0 to 4°, then centrifuged in a Servall centrifuge at 20,000 X g for 10 minutes at 4°, yielding the clear supernatant enzyme solution. The use of sand or ground glass with the mortar and pestle excludes the subsequent use of the glass homogenizer. Such a procedure is desirable for preparing extracts from fibrous roots, since the latter tends to crack the glass homogenizer. METHOD 3. Acetone-dried powders are prepared by placing the tissue, usually leaves, in a Waring blendor, covering with at least 5 vol. of chilled acetone, and blending vigorously for 30 seconds to 1 minute. The resulting slurry is filtered through a Btichner funnel, washed with an excess of chilled acetone, and the residue spread out on filter paper and allowed to dry at room temperature. The acetone powder, which is stored in a desiccator at 4 °, may be extracted with approximately 5 to 10 vol. of phosphate buffer essentially as described in method 2. Acetone powders have the advantage of being chlorophyll-free and tend to have a minimum of gum- and resin-like material which might otherwise interfere in subsequent fractionation of the extract. R. M. McCready and W. Z. Hassid, J. Am. Chem. Soc. 66, 560 (1944). a W. O. James and J. M. Cragg, New Phytologist 42, 28 (1943). 4 A. Nason, H. A. Oldewurtel, and L. M. Propst, Arch. Biochem. and Biophys. 38, 1 (1952). H. J. Evans and A. Nason, Plant Physiol. 28, 233 (1953).