1. Theoret. Biol. (1964) 6, 33-59 Principles of Tissue Fractionation CHRISTIAN DE DUVE Laboratory of Physiological Chemistry University of Louvain, Belgium and The Rockefeiler Institute, New York, N. Y., U.S.A. (Received 15 April 1963) This paper representsan attempt to develop logically the basic premises that tissuefractionation is : (a) a chemical method to be conducted according to the ground rules which govern chemical fractionation in general; (b) potentially applicable to the separationand characterization of all elements of cellular organization, whether known or unknown, which are not irretrievably lost in the initial grinding of the cells. It is shownthat the approach which best answersthese prerequisites is a purely analytical one, untrammeled by any preconceivedidea of the cyto- logical composition of the isolated fractions, and allowing their bio- chemicalproperties to be expressedas continuous functions of the physical parameterwhich determinesthe behaviour of subcellularcomponents in the fractionation systemchosen, Examples are given which illustrate the appli- cation of density gradient centrifugation in this type of approach, as well as the advantageswhich can be derived from the useof mediaof different composition. The resultsof suchexperiments are expressedin the form of distribution curves of biochemical constituents, as with other fractionation methods such as chromatography or electrophoresis,but with the differencethat the independent variable is related to a property, not of the constituent but of its host-particles. These curves can be taken to represent the mass distribution of the particles themselvesif the constituent is assumedto be homogeneouslydistributed amongstthem (postulateof biochemicalhomo- geneity). This assumptionhas been verified for a number of enzymes,which provide valuable markers to fix the position of their host-particleson the distribution diagrams. By comparing the distribution of an unlocalized constituent against the background of known distributions, especiallyunder a variety of experi- mental conditions, and by making use of all additional data which can be obtained by ancillary experiments,it is usually possibleeither to demon- strate the associationof the constituent with a known intracellular compo- nent, or to bring to light its possiblelocalization in an as yet unidentified type of particle. The existence,chemical properties, and structural features T.B. 33 3 34 CHRISTIAN DE DUVE of the latter can be further established when enough analytical resolution has been achieved to warrant a preparative attempt. Lysosomes as well as microbodies containing urate oxidase, catalase and D-amino acid oxidase have been identified in rat liver by following an approach of this kind. In the design of tissue fractionation experiments and in the interpretation of their results, it is essential to observe a rigorous logic and to employ an appropriately accurate vocabulary. The most important requirement in this respect is to maintain a strict distinction between the intracellular organelles or structures as they occur within the cells, the populations of particulate aggregates as they are present in the homogenate and react to the frac- tionation procedure, and the subcellular fractions as they are isolated and analysed. Introduction Numerous reviews have dealt with the theoretical basis and practical applica- tions of centrifugal fractionation. Most recent are that of Allfrey (1959), in which technical procedures are described and discussed in a particularly thorough fashion, and that of de Duve, Berthet & Beaufay (1959) which is specially devoted to density gradient centrifugation and includes a fairly complete mathematical treatment of the behaviour of a particle moving under the influence of centrifugal force in a homogeneous or in a heterogeneous medium. The object of the present paper is somewhat different and represents an attempt to outline what may be termed conceptual aspects of tissue fractiona- tion. The author and his associates began applying this technique shortly after it was first described and have used it more or less continuously ever since. The particular nature of the problems to which they became exposed, together with various results obtained by other investigators, led them to deviate progressively from the approach proposed by the originators of the method. As is often the case, the change was made empirically, largely by trial and error and with no clear understanding of the issues involved. A survey of the recent literature indicates that some workers have undergone a similar evolution, while many others still adhere to the original approach or, more exactly, to a somewhat distorted version of it. Looking back upon these investigations in the light of present knowledge, one can now recognize more easily the logical basis of the new approach as well as the nature of the premises from which it is derived. This paper is largely devoted to a retrospective analysis of this kind. Although written in the form of a personal essay, it is not intended to be dogmatic and is presented essentially in the hope that it will be of help to other workers engaged in tissue fractionation and will stimulate them to reflect more deeply on the significance and potentialities of this remarkable experimental tool. A shorter article on the same subject has already been published (de Duve, 1963). PRINCIPLES OF TISSUE FRACTIONATION 35 In writing this paper, it has been repeatedly necessary to distinguish clearly between two types of intracellular entities : the morphological compo- nents or organelles, such as the nuclei, the mitochondria and other intra- cellular particles, and the biochemical constituents, such as proteins, nucleic acids, enzymes, coenzymes, trace metals, and so on. In order to facilitate this distinction, the word component has been used consistently for the structural entities, and the term constituent for the biochemical ones. In line with this convention, one of the main objects of cytochemistry may be defined as the structural localization of cell constituents, or alternatively, as the biochemical characterization of cell components. Centrifuge and Microscope Cytochemistry Though not originally developed by biochemists, tissue fractionation is fundamentally a chemical method. It may be defined as the chemist’s approach to an integrated study of cell structure and function, as opposed to that of the morphologist, cytochemical visualization. While both methods are directed primarily towards the correlation of biochemical events with subcellular structures, the manner in which this common aim is pursued is charac- teristically influenced in each case by the biases of the parent discipline. In visual cytochemistry, the main concern is to render biochemical con- stituents and enzymes visible within the familiar framework of tissue or cell architecture. Preservation of the latter is perforce a dominant factor and is achieved at the cost of variable but usually fairly high losses in biochemical precision. On the other hand, fractionation methods follow the classical steps of chemical separation and analysis, and sacrifice a great deal of morpho- logical information to their basic requirements for biochemical integrity and analytical accuracy. Both approaches fall short of the high standards adopted in the more classical forms of biochemistry or cytology, and it is therefore not sur- prising that they have inspired a number of pessimistic pronouncements, usually emanating from one side and directed at the other. In general, the view that an enzyme may emerge unscathed from the usual fixation procedures and will later avoid a number of kinetic traps and conveniently surround itself with the artificially insolubilized products of its own action, is as abhorrent to the enzyme chemist as is to the morphologist the concept that an intact nucleus or mitochondrion may somehow miraculously be separated with a centrfuge from the remains of chaotically dismembered tissues. Such criticisms, though useful in stimulating a constant search for improve- ment, leave out the fact that the standards of biochemistry and cytology have been raised to their present degree of eminence mostly by a process of mutual exclusion, in which structural and biochemical integrity have been deliberately 3--z 36 CHRISTIAN DE DUVE sacrificed to one another. To condemn any correlative approach a priori on the basis of a single set of criteria is actually to deny the possibility of such an approach, which of necessity must rest on a compromise. It is to forget that with all its defects, the correlative attempt must bring the investigator in closer contact with the true properties of the living cell, in which structure and function are undissociable. A more constructive attitude is that adopted by many workers in both fields, who consider the two approaches as comple- mentary rather than opposed, and the artifacts involved as factors to be clearly recognized, minimized by all possible means and taken into account, when unavoidable, in a realistic interpretation of the results. In practice, owing to a fundamental difference between the two techniques, they have so far covered little common ground and rendered unequal services. In visual cytochemistry, especially when directed at the intracellular localiza- tion of enzymes, the main limitation lies in the enzymes themselves, of which only a few have so far lent themselves to appropriate staining procedures. In addition, years of arduous work may be necessary