The Influence of MacromolecularCrowding and MacromolecularConfinement on BiochemicalReactions in PhysiologicalMedia*Published, JBC Papers in Press, February 15, 2001, DOI10.1074/jbc.R100005200Allen P. Minton‡From the Section on Physical Biochemistry, Laboratoryof Biochemistry and Genetics, NIDDK, NationalInstitutes of Health, Bethesda, Maryland 20892Detailed knowledge of the rates, equilibria, and mechanismof biochemical reactions has traditionally been acquiredthrough experiments conducted on solutions containing lowconcentrations (less than about 1 mg/ml) of total protein, nu-cleic acid, and/or polysaccharide together with buffer salts, lowmolecular weight substrates, and cofactors as required. In con-trast, biochemical reactions in living systems take place inmedia containing substantially greater total concentrations(50–400 mg/ml) of macromolecules that may be present insolution and/or in indefinitely large arrays (e.g. cytoskeletalfibers) (1, 2). Because no single macromolecular species may bepresent at high concentration, but all species taken togetheroccupy a significant fraction of the volume of the medium, suchmedia are referred to as “crowded” (3) and/or “confining” (4)rather than “concentrated,” depending upon whether the mac-rosolutes are soluble and/or structured. Fig. 1 provides a sche-matic illustration of crowding and confinement in eukaryoticcytoplasm. In such media, nonspecific interactions betweenmacrosolutes contribute significantly to the total free energy ofthe medium. High concentrations of “background” macromole-cules that do not participate directly in a particular test reac-tion have been observed to induce order-of-magnitude orgreater changes in the rates and equilibria of numerous testreactions (see below). To properly assess the physiological roleof a particular reaction or set of reactions characterized invitro, it is important to consider the possible influence of crowd-ing and/or confinement upon the reaction in its physiologicalmilieu.Nonspecific InteractionA nonspecific interaction between a pair of macromoleculesdoes not depend strongly upon details of the primary, second-ary, or tertiary structure(s) of the interacting macromoleculesbut rather upon global properties such as net charge, dipole ormultipole moment, the polarity of surface residues, and mac-romolecular “shape.” Nonspecific interactions may be eitherrepulsive (steric, electrostatic) or attractive (electrostatic, hy-drophobic) and are generally substantially weaker on a pair-wise basis than specific interactions between reaction partners.The concept of “nonspecific interaction” is widely misunder-stood. Many if not most biomedical researchers still regardsuch interaction as an artifact of a particular experimentalsystem that interferes with the acquisition of meaningful data.Strategies such as extrapolation of results to zero macromolec-ular concentration are devised for the reduction or eliminationof the influence of nonspecific interaction on a test reaction.Although such procedures may be appropriate in certain spe-cific experimental situations, they do not necessarily provideresults that are more meaningful in a biological context. On thecontrary, significant nonspecific interaction is an unavoidableconsequence of crowding and confinement in most or all phys-iological fluid media. To understand molecular processes insuch media one must therefore take account of nonspecificinteractions rather than attempt to eliminate them.Effect of Nonspecific Solute-Solute Interaction uponChemical EquilibriaThe contribution of a particular solute species X to the totalfree energy of the system is a function of an effective concen-tration, called the thermodynamic activity of X, denoted by ax.Thermodynamics teaches that equilibrium constants are gen-erally expressed in terms of equilibrium activities rather thanactual concentrations. As a simple example, consider a proteinmolecule that may reversibly self-associate to form a dimer.The equilibrium association constant for this reaction is K12o⫽(a2/a12), where subscripts 1 and 2 refer to monomer and dimer,respectively. Biochemists are accustomed to seeing equilibriumconstants written as ratios of equilibrium concentrations.However, the so-called equilibrium constant written in termsof concentrations, K12, is actually an apparent constant re-lated to the true equilibrium constant, K12o,byK12⬅ (c2/c12) ⫽K12o(␥12/␥2), where␥idenotes the ratio of effective to actualconcentrations of species i, termed the activity coefficient. Theactivity coefficient has a precise definition in terms of nonspe-cific solute-solute interaction, ln␥i⫽⬍gi⬎/kT, where ⬍gi⬎denotes the (composition-dependent) equilibrium average freeenergy of nonspecific interaction between a molecule of speciesi and all of the other macrosolutes present in the medium, k isthe Boltzmann constant, and T is the absolute temperature.Excluded and Available VolumeSteric repulsion is the most fundamental of all interactionsbetween macromolecules in solution and is always present atfinite concentration, independent of the magnitude of addi-tional electrostatic or hydrophobic interactions. Because solutemolecules are mutually impenetrable, the presence of a signif-icant volume fraction of macromolecules in the medium placesconstraints on the placement of an additional molecule of testmacrosolute that depend upon the relative sizes, shapes, andconcentrations of all macrosolutes in the medium. Fig. 2 depictsa region, demarcated by a square outline, in a solution contain-ing spherical “background” macrosolutes of radius rb, coloredblack, that occupy ⬃30% of the total volume (vtot) of the spec-ified region. The available volume (va,T) is defined to be thatpart of the volume of the region which may be occupied by thecenter of mass of a molecule of a spherical test species T ofradius rtadded to the solution. If the test species is very smallrelative to the background species (Fig. 2A), then the availablevolume, indicated in blue, is approximately equal to that part ofthe total volume not occupied by the background species, i.e.⬃0.7 vtot. However, if the size of the test species is comparable* This minireview will be reprinted in the 2001 Minireview Compen-dium, which will be available in December, 2001.‡ To whom correspondence should be addressed: Bldg. 8, Rm. 226,NIH, Bethesda, MD 20892-0830. Tel.: 301-496-3604; Fax: 301-402-0240; E-mail:
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