We describe an approach to exploring cell surface-cytoskeleton interactions through direct measurements of the mechanical resistance of living cells to locally applied forces. These measurements are sensitive to variations in structure across the cell and at various depths below its surface. We find that local cellular deformability depends on the temperature and on the integrity of the cytoskeleton. Cytochalasin B increases the deformability of all regions of the cell except the nucleus.

Wild-type vesicular stomatitis virus and temp.-sensitive mutants with lesions in the M protein were compared under conditions in which the mutant M protein was functional. All 4 different temp.-sensitive mutants exhibited a coordinated increase in assocn. of M protein with membrane (or viral envelope) and a decrease in assocn. with nucleocapsid as shown by labeling with [115I]iodoaphthylazide, fluorescence photobleaching studies of infected cells, nonreductive dissocn. in SDS, and disruption with nonionic detergents. The rate of viral budding decreased in temp.-sensitive mutant-infected cells, suggesting that coordinate changes in affinities of the mutant M protein give rise to a different rate limiting step for budding. The surface G glycoprotein of wild-type virus-infected cells was apparently not predominantly assocd. with protein M; in mutant-infected cells, much more of the surface G was M-assocd. and immobilized.

We have studied the lateral mobility and distribution of beta receptors on Chang human liver cells by fluorescence photobleaching recovery and video intensification microscopy. The beta receptors were labeled with the fluorescent antagonist 7-(2-allylphenoxy)-2,2-dimethyl-6-hydroxy-1-(4-nitrobenzo-2-oxa-1,3-diazolyl)-1 ,4-diazaheptane (Alp-NBD). Sixty to 75% of the staining was specific (displaceable by unlabeled antagonists). Most of the antagonist-occupied beta receptors were immobile, because only 15-25% of their fluorescence recovered on the experimental time scale at 23 degrees C. This immobility correlates with the clustered distribution of Alp-NBD--beta-receptor complexes at 4 degrees C and 37 degrees C. The beta receptors appear to be aggregated prior to antagonist binding, because visible patches were observed immediately after labeling for 30 sec at 4 degrees C. Preincubation at 37 degrees C with (--)-isoproterenol, a beta agonist, prior to Alp-NBD labeling induced a time-dependent release of the beta receptors to a more homogeneous distribution and increased the mobile fraction to 70-80% (lateral diffusion coefficient = 1.4 X 10(-9) cm2/sec at 23 degrees C). This is not due to an effect on membrane fluidity, because the diffusion coefficient of a lipid probe was not altered. The time course of agonist-induced beta-receptor mobilization correlates with receptor loss and adenylate cyclase desensitization but is much slower than adenylate cyclase activation. This indicates that adenylate cyclase activation by beta receptors does not require macroscopic lateral mobility of the majority of the beta receptors.

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.

Temp.-sensitive M-protein mutants of vesicular stomatitis virus (complementation group III) were characterized with regard to their assocn. with nucleocapsids and with membranes. Salt dependence of the inhibition of RNA synthesis by disrupted virions showed that the M-protein of all 4 mutants studied had a decreased functional, electrostatistically stabilized assocn. with nucleocapsid. Fluorescence photo-bleaching measurements showed that mutant M-protein in infected cells immobilized G-protein on the cell surface to a much larger extent with infected cell membranes than did the wild-type (wt), suggesting that mutant M-protein increased to a greater extent with infected cell membranes than did wt M-protein. The labeling of purified virions with the hydrophobic photoactive probe iodonaphthylazide showed that the M-protein of mutant virions was labeled 2-3-fold more strongly than was the M-protein of wt virions, also suggesting greater membrane assocn. for mutant as opposed to wt M-protein. These coordinate effects, exhibited by all the M-protein mutants studied, are interpreted in terms of a single mutational change affecting only 1 of 2 independent binding sites. This interpretation provides the basis for a provisional kinetic scheme for budding of virions from the plasma membranes of infected cells.

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. This immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed.

Lateral mobilities of fluorescent cell surface probes were measured on normal- (3T3) and transformed- (SV3T3) cultured mouse fibroblast. There is little discernible difference in the mobilities of a lipid analog (dioctadecylindocarbocyanine iodide) a fluorescein deriv. of ganglioside GM1, and tetramethylrhodamine-labeled succinylated concanavalin A. The 2 cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated by increased overall membrane fluidity but are assocd. with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivs. and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.

The lateral mobilities of membrane lipids and proteins were detd. during the cell cycle of synchronized C1300 mouse neuroblastoma cells. The diffusion coeff. of lipids reached a min. in mitosis, increased 2- to 3-fold during G1 remained const. at maximal values during S, and decreased again shortly before mitosis. Membrane proteins also exhibited min. diffusion coeffs. in mitosis, followed by a similar rise in G1. However, as cells proceeded through S and G2, the lateral mobility of the membrane proteins gradually decreased. Apparently, lipid mobility is controlled by the fluidity of the membrane lipid matrix, whereas protein mobility is governed also by other constraints.

Lateral diffusion of membrane lipids and proteins was detd. in differentiating C1300 mouse neuroblastoma cells by fluorescence photobleaching recovery measurements. During differentiation, the lateral diffusion of membrane lipids and proteins increased in the extending neurites. Thus, topog. heterogeneity appears in the cell membrane, whereby more fluid domains are located in the membrane of the neurites.

Fluorescence correlation spectroscopy is extended to systems that undergo translation or laminar flow in a sample cell. Theor. and exptl. results and uniform and nonuniform velocity profiles are included. Concn. correlation anal. exts. microscopic rate parameters from measurements of the spntaneous concn. fluctuations, which occur even at equil. Fluorescence is one of the most sensitive means of monitoring these fluctuations. Anal. of flowing or translating systems (1) offers a method of measuring no. concns. of selected species, for example, of aggregates of polymers, (2) provides a nonperturbing velocity probe, (3) sometimes allows one to circumvent photolytic degrdn., (4) has proved extremely helpful in testing and aligning app. for fluorescence correlation measurements and in verifying theor. analyses, and (5) may be required for interpretation of results obtained on systems in motion, even though that motion is undesired or initially unsuspected. Both theor. and exptl. results are included for combined Poiseuille flow and diffusion in the geometry which is of most practical interest. Theor. expressions for the much simpler cases of nondiffusive Poiseuille flow as well as uniform flow or translation with or without difusion constitute limiting cases which are displayed explicitly.

Diffusion coeffs. (D) of a lipid probe and labeled proteins in L-6 myoblast membranes were measured giving D (protein) = .apprx.2 .times. 10-10 cm2/s and D (lipid probe) = .apprx.9 .times. 10-9 cm2/s. Some of the membrane proteins were immobile, but the lipid probe diffused freely over macroscopic distances. Cytochalasin B slowed protein but not lipid probe diffusion.

The fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, 2 states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coeff. of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion const. of mobile receptors was partially reversed by treatment with microtubule-disrupting agents, such as Colcemid and Vinca alkaloids. High doses of sol. Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was not obsd. These exts. provide addnl. support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the mol. level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.

Fluorescence photobleaching recovery and immunofluorescence methods were used to study the lateral mobility and topog. distribution of a major cell surface glycoprotein (CSP) from fibroblasts. Both endogeneous CSP and fluorescent-labeled exogeneous CSP were bound to the cell surface in fibrillar pattern and were immobile on the exptl. time scale. Azide, vinblastine, and cytochalasin B did not alter the immobility and cell surface distribution of the CSP mols. Therefore, oxidative phosphorylation and the cytoskeleton are not responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils did, however, impede the diffusion of a lipid probe, a ganglioside analog, or various surface antigens. Therefore, the fibrils apparently do not form a barrier across the lipid phase of the plasma membrane. In contrast, concanavalin A was bound to CSP and was largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that the other types of immobile concanavalin A receptors also exist. CSP did not bind to lipid bilayers composed of lecithin or oxidized cholesterol. It did bind to dextran-coated bilayers as a diffuse distribution of mobile mols. that can patch after addn. of antibodies to CSP. Apparently, CSP mols. do not interact strongly with other CSP mols. under these latter conditions. Exogenous CSP was bound to regions on the cell surface that already contained CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addn. to CSP.

A discussion and review with 13 refs. of recent exptl. evidence concerning the valence, mobility, and membrane integration of the IgE receptor of mast cells and basophils and its relation to the triggering of the degranulation process.

Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coeffs. of fluorescent lipid analogs in lipid bilayer membranes indicated self-diffusion coeffs. D .gtorsim. 10-7 cm2/s for various lipid systems above their reported transition temps. Cholesterol in egg lecithin at a mol ratio of 1:2 reduced D by .apprx.2-fold; retained hydrocarbon solvent increased it by 2- to 3-fold.

The lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat was studied. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions effective diffusion coeffs. (D) of the receptor complexes in the range 8 .times. 10-12 .ltoreq. D .ltoreq. 3 .times. 10-11 cm2/sec were obsd. which is 2 orders of magnitude lower than was found for a fluorescent lipid probe, D .simeq.8 .times. 10-9 cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities were not influenced by azide, colchicine, or preincubation at low temp. Cytochalasin B and low temps., during the time of measurement, decrease the lateral transport rate.

An effective exptl. system was developed for the characterization of mol. and structural mobility that incorporates a modified fluorescence microscope geometry and a variety of anal. techniques to measure effective diffusion coeffs. from <10-11 cm2/s to >10-6 cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are used. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochem. in a microscopic vol. by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concn. fluctuations in microscopic sample vols. The kinetics are analyzed by computing time correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solns., lipid bilayer membranes, and mammalian cell plasma membranes.

This paper describes the principles and recent applications of two new methods for measuring rates of macroscopic lateral transport of fluorescent-labeled particles on the surface of individual cells. Both methods are based on microfluorimetric measurements of fluorescence intensity from a small open region (greater than or equal to 1 micronm radius) on the cell surface. Transport rates are measured from the rates at which the measured fluorescence intensity changes due to entrance or departure of fluorophores from the region. One method, "Fluorescence Photobleach Recovery" (FPR), uses a brief intense pulse of light to create an initial concentration gradient over the spot by irreversible photochemical destruction of fluorophores. The rate of fluorescence recovery due to transport of unbleached fluorophores into the observation region is the primary experimental datum. The other method, "Fluorescence Correlation Spectroscopy" (FCS), is based on a statistical analysis of spontaneous fluctuations of numbers of fluorophores in the observation region and does not require a perturbation to generate an initial concentration gradient. FCS is mainly useful to measure relatively fast processes (D greater than or equal to 10(-9) cm 2/sec) in stable systems; FPR can be used to measure both slow and fast transport in less stable systems. Using both FCS and FPR, the diffusion coefficient of a fluorescent lipid probe in rat myoblast plasma membranes was measured to be D = (9 +/- 4) X 10(-9) cm2/sec over a range of at least 4 micronm. FPR was used to measure the lateral mobility of the fluorescent labeled lectin concanavalin A complexed to myoblast plasma membrane "receptors." A fraction of the complexes were immobile on the time scale of the experiment (D less than 8 x 10(-12) cm2/sec). The remainder of the complexes had effective diffusion coefficients far smaller than expected from the measurements on the lipid probe (8 x 10(-12) less than or equal to D less than or equal to 3.3 x 10(-11) cm2/sec). Moreover the mobility depended on the valence, dose, and time of occupancy of the lectin on the membrane, suggesting that an aggregation of complexes was occurring during the experiment. Cytochalasin B decreased the mobility of complexes, suggesting an influence of microfilaments on the transport process. Neither azide nor colchine affected measured transport rates. These results indicate the operation of constraints on the mobility of the lectin-receptor complexes beyond that exerted by the viscous resistance of the lipid bilayer membrane matrix. The two types of interactions revealed by our current experiments - interactions of the complexes with microfilaments and with each other (aggregation) - seem insufficient to account entirely for the low observed mobility.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring 2-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled mols. in .apprx.10 .mu.m2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. The theor. basis and some practical guidelines are presented for simple, rigorous anal. of FPR expts. Information obtainable from FPR expts. includes: identification of transport process type, i.e., the admixt. of random diffusion and uniform directed flow; detn. of the abs. mobility coeff., i.e., the diffusion const. and (or) flow velocity; and the fraction of total fluorophore that is mobile. To illustrate the exptl. method and to verify the theory for diffusion, exptl. models are described comprising aq. solns. of rhodamine 6G.

Direct, quant. measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture revealed 2 coexisting distinct classes of AChR: (1) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (2) immobile, dense, highly granular AChR in patches of 10-60 .mu.m size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addn., evidence of turnover of AChR labeled with tetramethylrhodamina-.alpha.-bungarotoxin was seen in the gradual internalization of surface fluorescence within 36 h after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

A review with 31 refs. The topics covered include: theory, exptl. arrangements and results of fluorescence correlation spectroscopy (FCS), light scattering expts. as the indicator of occupation no. fluctuations, concn. correlation spectroscopy in rotational diffusion and flow anal., conductometric indication of occupation no. fluctuations, and problems and prospects.

A new relaxation method was developed for measuring the kinetics of chem. reactions perturbed very slightly from equil. by a small change in pressure. The pressure was applied by compressing a soln. using the voltage-dependent extension of a piezoelec. crystal stack. Reaction progress was followed by monitoring chem. sp. optical properties such as absorbance, fluorescence, or light scattering. Examples were given of using the method to study the relaxation times of several chem. systems that have been studied well by the temp.-jump method (Ni(II)-.alpha.-alanine complex formation, BeSO4 system). The method should be esp. useful in studying the dynamic aspects of conformational transformations of biol. macromols. and of highly cooperative processes.

This paper describes the first exptl. application of fluorescence correlation spectroscopy, a new method for detg. chem. kinetic consts. and diffusion coeffs. These quantities are measured by observing the time behavior of the tiny concn. fluctuations which occur spontaneously in the reaction system even when it is in equil. The equil. of the system is not disturbed during the expt. The diffusion coeffs. and chem. rate consts. which det. the av. time behavior of these spontaneous fluctuations are the same as those sought by more conventional methods including temp.-jump or other perturbation techniques. The expt. consists essentially in measuring the variation with time of the no. of mols. of specified reactants in a defined open vol. of soln. The concn. of a reactant is measured by its fluorescence; the sample vol. is defined by a focused laser beam which excites the fluorescence. The fluorescent emission fluctuates in proportion with the changes in the no. of fluorescent mols. as they diffuse into and out of the sample vol. and as they are created or eliminated by the chem. reactions. The no. of these reactant mols. must be small to permit detection of the concn. fluctuations. Hence the sample vol. is small (10-8 ml) and the concn. of the solutes is low (.apprx.10-9 M). We have applied this technique to the study of 2 prototype systems: the simple example of pure diffusion of a single fluorescent species, rhodamine 6G, and the more interesting but more challenging example of the reaction of macromol. DNA with the drug ethidium bromide to form a fluorescent complex. The increase of the fluorescence of the ethidium bromide upon formation of the complex permits the observation of the decay of concn. fluctuations via the chem. reaction and consequently the detn. of chem. rate consts.

We describe a method for detg. chem. kinetic consts. and diffusion coeffs. by measuring the rates of decay of spontaneous concn. fluctuations. The equil. of the system is not disturbed during the measurement. We measure the no. of mols. of a specified type in a defined open vol. as a function of time and compute the time course of the deviations from the thermodn. mean concn. The method is based on the principle that the rates of decay of spontaneous microscopic fluctuations are detd. by the same phenomenological rate coeffs. as those of macroscopic departures from equil. which result from external perturbations. Hence, an anal. of fluctuations yields the same chem. rate consts. and diffusion coeffs. as are measured by conventional procedures. In practice the no. of the specified mols. is measured by a property such as absorbance or fluorescence which is specific and sensitive to chem. change. The sample vol. is defined by a light beam which transverses the cell. As the mols. appear in or disappear from the light beam, either due to diffusion or chem. reaction, their concn. fluctuations give rise to corresponding fluctuations of the intensity of adsorbed or emitted light. This paper presents the theory needed to derive chem. rate consts. and diffusion coeffs. from these fluctuations in light intensity. The theory is applied to 3 examples of general interest: pure diffusion in the absence of chem. reaction; the binding of a small rapidly diffusing ligand to a larger slowly diffusing macromol.; and a unimol. isomerization. The method should be especially useful in studying highly cooperative systems, relatively noncooperative systems with intermediate states closely spaced in free energy, small systems, and systems not readily subject to perturbations of state.

Correlations of thermodynamic concn. fluctuations were measured in a chem. reactive system at equil. by observing fluctuations of the fluorescenceof a reaction producct. The expt. yields the chem. rate consts. and diffusion coeffs. and shows the coupling among them. Data are reported for binding of ethidium bromide to DNA.

A summary is presented of a procedure for a complete kinetic anal. of a reaction scheme. The anal. is applied to an interpretation and assessment of a steady-state approximation. A complete soln. of the kinetic scheme yields the concn. as a function of time of each of the conformational species. Ref.

Temp.-jump studies have been used to detect a rapid reaction in the thermal unfolding of RNase A. The fast reaction occurs over a wide range of pH, and the results of a detailed study at pH 1.3 are reported here. Although its amplitude is small, the reaction is easily measurable over the entire temp. range of thermal unfolding. It occurs in the msec time range, and is faster by 3-4 orders of magnitude than the slow unfolding reaction studied previously. Unfolding is measured here by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. Since the fast reaction has a detectable amplitude only in the temp. range of unfolding, it apparently detects the presence of intermediate, partly-folde states. Previous equil. studies of the unfolding of RNase A in the pH range 1-2 have indicated that it is essentially a 2-state reaction, without detectable intermediates. The existence of a rapid transient phase in the unfolding of RNase A had been predicted previously from a model for this unfolding reaction, based on nucleation-dependent sequential folding. The model served to reconcile kinetic and equil. studies of the thermal unfolding reaction of RNase A at neutral pH. Kinetic studies had shown that the slow unfolding reaction, measured at 287 nm, could be represented as a single exponential process, as expected for a 2-state reaction. However, earlier equil. measurements, esp. the calorimetric studies of Sturtevant and coworkers, had revealed significant deviations from the 2-state behavior at neutral pH. These conflicting observations are explained by the model, which satisfies closely many criteria for a 2-state unfolding, even when appreciable concns. of partly folded mols. are present. In particular, it predicts that the final, and major, portion of the kinetic reaction will occur as a single process characterized by an exponential time course.

The hydrolysis of 2-methyl-2-thiazoline was studied as a function of pH in acid and alk. solns. The reaction rate, as determined by ultraviolet absorption at 260 m.mu. and by Raman spectroscopy, indicated a max. at pH 3. This max. was explained by the formation of an intermediate hydroxythiazolidine from the acidic thiazoline. The hydroxythiazolidine then gave both the N-and S-acetyl-.beta.-mercaptoethylamine. Rate consts. for each step were determined. The cyclization of N-acetyl-.beta.-mercaptoethylamine to produce the thiazole ring was also investigated.