1. Field Of The Invention.
This invention relates to a method for the chemical purification of enzymatic proteins. Known chemical separation methods for enzymatic proteins are usually based upon differential precipitation of the preferred enzyme from other undesirable enzymes and impurities which contaminate the mixture. Where an enzyme is not separable from other impurities found in the enzyme mixture, in many cases this renders it useless in particular applications. For example, the enzyme, glucose oxidase, is usually found in the presence of the enzyme catalase. Glucose oxidase contains a carbohydrate moiety and is thus a carbohydrate containing enzyme. Glucose oxidase catalyzes the reaction of glucose to form gluconic acid and hydrogen peroxide. Catalase catalyzes the degradation of hydrogen peroxide. In many applications glucose oxidase is used to generate hydrogen peroxide to quantify the glucose substrate. When catalase is present the hydrogen peroxide generated by the glucose oxidase is partially destroyed. The method is thus less accurate and in many cases totally ineffective. Other examples are known where one enzyme is extremely difficult to separate from another and this inability to separate the enzymes limits the utility of the preferred enzyme.
Additionally, presently known methods of separating enzymes are time consuming, possibly on the order of hours, usually require a number of different reagents to complete the separations process, and result in a low yield of the separated preferred enzyme. These drawbacks in the cases where separation is possible as well as the circumstance where separation is not completely possible, or possible only with very undesirable levels of contaminants, are overcome by the present invention.
2. Description Of The Prior Art.
Most of the known prior art relating to the chemical separation of enzymes from other enzymes or other chemical impurities in a mixture are concerned primarily with three methods of separation. The first method is differential precipitation, using water-miscible solvents. U.S. Pat. No. 3,616,232 discloses the use of water-miscible alkanols, alkylketones, and cyclic ethers as precipitating agents to separate proteins. The solution containing the proteins is mixed with the water-miscible solvents, in which the enzymes themselves are not soluble, in a particular volume to volume fraction. After the solvents are added, the mixture is shaken, and at particular volume to volume fractions different proteins fractionate from the mixture. Similarly, U.S. Pat. Nos. 3,645,851 and 2,926,122 disclose the use of low molecular weight alcohols to differentially precipitate proteins from an aqueous solution, and then collect the precipitated proteins. Most of these differential solvent precipitation methods require precise balancing of solvent volumes, solution temperatures, and the time the solutions are allowed to stand. These methods always involve the inherent risk of solvent denaturation of the enzyme or protein which is preferred and is being recovered. Additionally, these methods usually take long periods of time and in many cases have low yields of the preferred enzyme.
The second commonly used separation method of enzyme or protein purifcation involves differential precipitation without the use of solvents. In these methods, generally speaking, an inorganic salt or organic base is added to a solution of the preferred enzyme. The preferred enzyme or the contaminating enzymes are differentially precipitated from the solution. The precipitate or the supernatant liquid, whichever contains the preferred enzyme, is then processed by known chemical methods, for example, gel filtration. These methods also usually require long periods of time, the separation and handling of precipitates, and generally use a number of reagents. For example, U.S. Pat. No. 3,930,953 discloses the precipitation of glucose oxidase from an aqueous solution by mixing the glucose oxidase solution with diaminoethoxyacridine lactate. The lactate forms a precipitate with the glucose oxidase. The solution is allowed to stand for as long as twenty-four hours at low temperatures, whereupon the lactate-protein complex precipitates out of solution. The complex is then recovered and destroyed by the addition of large quantities of chloride salts and the resultant free glucose oxidase is separated by conventional chemical methods. Similarly with U.S. Pat. Nos. 3,265,587 and 3,269,918 the desired protein is precipitated or the impurities are precipitated and the fraction containing the desired protein is further processed by conventional chemical methods. Typical precipitating reagents are calcium, barium, and ammonium sulfates.
The third type of known separation method comprises contacting the enzyme mixture with a solution containing an insolublized coenzyme, which is reacted with respect to one or more of the enzymes in the mixture. The enzyme in the mixture becomes attached to the insolubilization support through the coenzyme. The support is removed from the mixture with the enzyme attached thereto, and the enzyme is eluted from the support for further processing.
None of the prior art discussed above discloses a method for the separation of carbohydrate containing proteins wherein the carbohydrate portion of the carbohydrate containing preferred enzyme is modified. The process, according to the present invention, is very rapid compared to methods known in the art. Also, the method is relatively inexpensive and has a very high yield of the preferred enzyme. In many cases, substantially all of the preferred enzyme is isolated from impurities, with yields of the preferred enzyme on the order of 90% recovery and above.
Additional references to precipitation methods of purification of proteins of enzymatic types are: "The Oxidation of Glucose and Related Compounds by Glucose Oxidase from Aspergillus niger". John H. Pazur and Kjell Kleppe, Biochemistry, Vol. 3, pp. 578-83 (1964); "Purification and Properties of the Glucose Oxidase from Aspergillus niger" Bennett E. P. Swoboda and Vincent Massey, Journal of Biological Chemistry, Vol. 240, pp. 2209-15 (1965); "Comparative Studies on the Glucose Oxidase of Aspergillus niger and Penicelium amagasakiense" Satoshi Nakamura and Smiko Fujiki, Journal of Biochemistry, Vol. 63, pp. 51-8 (1968); and "The Glucose Oxidase Mechanism, Interpretation of the pH Dependence", Michael K. Weibel and Harold J. Bright, Journal of Biological Chemistry, Vol. 246, pp. 2734-44 (1971).
It has been recognized that carbohydrate moieties on carbohydrate containing enzymes can be treated with oxidizing agents preparatory to immobilization by covalent bonding to supports. See, "The Immobilization of Glucose Oxidase. Activation of its Carbohydrate Residues", O. R. Zaborsky and J. Ogletree, Biochem. Biophys. Res. Commun., Vol. 61, pp. 210-16 (1974).
Also, the oxidation of carbohydrate moities on proteins has been accomplished in order to determine kinetic and structural properties of proteins in solution. For examples of such determinations see, "A Role of the Carbohydrate Moiety of Glucose Oxidase: Kinetic Evidence for Protection of the Enzyme from Thermal Inactivation in the Presence of Sodium Dodecyl Sulfate", S. Kakamura and S. Hayashi, FEBS Letters, Vol. 41, pp. 327-9 (1974), and "The Composition and Structure of Carbohydrate Moiety of Stem Bromelain", Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, Vol. 9, pp. 25-32 (1970).
Based on the above discussed art, no method is believed known for the separation of a carbohydrate containing enzymatically active protein from a contaminating protein or enzyme by the modification of the carbohydrate moiety according to the method of the present invention.