Increasing Flash Intensity Research Articles

Quantitative Assessment of the Canine Pupillary Light Reflex

To develop instrumentation and methods for thorough quantitative assessment of the pupillary light reflex (PLR) in dogs under varying stimulus conditions. The PLR was recorded in normal Dachshunds using a custom system allowing full user control over stimulus intensity, color, and duration. Chemical restraint protocols were compared to determine which protocol provided for optimal baseline stability of pupil size and appropriate eye positioning. A series of white light stimuli of increasing intensity was used to elicit pupil constriction. Pupil images were concurrently recorded using continuous infrared illumination and an infrared-sensitive camera. The PLR was also recorded in response to blue and red stimuli. With injectable chemical restraint alone, spontaneous fluctuations in pupil size occurred independent of light stimulation, and spontaneous eye movements made it difficult to fully visualize the pupil. Combined injectable chemical and inhalation restraint provided a steady baseline pupil size throughout PLR assessment and allowed for stable positioning of the eye using a conjunctival stay suture. Robust PLRs were elicited with all light colors. PLR constriction amplitude increased with increasing flash intensity and ranged from 5% to 70%. A recording system and protocol have been developed to reliably quantify the canine PLR. The techniques and instrumentation will be useful for objective quantitative assessment of the PLR in dogs and other species in research applications and may be useful in clinical veterinary ophthalmology and neurology if PLR abnormalities detected with these procedures can be associated with specific diseases.

Cone Phosphodiesterase-6 ' Restores Rod Function and Confers Distinct Physiological Properties in the Rod Phosphodiesterase-6 -Deficient rd10 Mouse

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the vertebrate phototransduction pathway in rods and cones. Rod PDE6 catalytic core is composed of two distinct subunits, PDE6α and PDE6β, whereas two identical PDE6α' subunits form the cone PDE6 catalytic core. It is not known whether this difference in PDE6 catalytic subunit identity contributes to the functional differences between rods and cones. To address this question, we expressed cone PDE6α' in the photoreceptor cells of the retinal degeneration 10 (rd10) mouse that carries a mutation in rod PDEβ subunit. We show that adeno-associated virus-mediated subretinal delivery of PDE6α' rescues rod electroretinogram responses and preserves retinal structure, indicating that cone PDE6α' can couple effectively to the rod phototransduction pathway. We also show that restoration of light sensitivity in rd10 rods is attributable to assembly of PDE6α' with rod PDE6γ. Single-cell recordings revealed that, surprisingly, rods expressing cone PDE6α' are twofold more sensitive to light than wild-type rods, most likely because of the slower shutoff of their light responses. Unlike in wild-type rods, the response kinetics in PDE6α'-treated rd10 rods accelerated with increasing flash intensity, indicating a possible direct feedback modulation of cone PDE6α' activity. Together, these results demonstrate that cone PDE6α' can functionally substitute for rod PDEαβ in vivo, conferring treated rods with distinct physiological properties.

Combined effects of aging and a pro‐diabetic diet on retinal function in a murine model of aging of the human eye

Abstract Purpose Improvement of life expectancy and changes in the dietary behaviour of developed populations are accompanied with the prevalence of diabetes and age‐related ocular pathologies. Aging of the retina is characterized by accumulation of lipids at its basement. Meanwhile increased oxidative stress is one of the features of aging and diabetes. The goal of our study was to evaluate the effects of a pro‐diabetic fructose‐rich diet on the functioning of the retina in a murine model of aging of the human eye, the ApoB100,LDLR‐/‐ mice. Methods 4 month‐old ApoB100,LDLR‐/‐ and SF2J control mice were bred under either a standard chow or a fructose‐rich diet (60%) during 6 months (n=6 in each group). The functionality of the retina was further evaluated by single flash electroretinography (ERG) under scotopic conditions to assess the global response of the retina. Flicker ERG was monitored by increasing flash intensity upon 10Hz‐fixed frequency of the stimulus to specifically assess the sensitivity of rods and cones. Results The fructose‐rich diet significantly decreased a‐ and b‐wave amplitudes of the retina to single flash ERG. This diminution was intensified in ApoB100, LDLR‐/‐ mice compared to control mice: ‐55.5% vs ‐33.4% for the a‐wave, ‐41% vs ‐38% for b‐wave, respectively. The fructose‐enriched diet impaired the sensitivity of rods by 0.3 log unit in both strains of mice. Conclusion Oxidative stress triggered by a pro‐diabetic diet promotes the deleterious effects of aging on the retina by reducing its functionality and impairing its sensitivity to light stimulus.

Attenuation of oscillatory potentials in nob2 mice

To examine changes in inner retinal function of nob2 mice, expressing a null mutation in Cacna1f encoding the Ca(V)1.4 subunit of voltage-dependent calcium channels. CACNA1F mutations underlie one form of incomplete X-linked congenital stationary night blindness (CSNB2). In addition to a loss of dark-adapted (rod-driven) visual sensitivity, electroretinogram (ERG) b-waves and oscillatory potentials (OPs) are decreased in CSNB2 patients. ERGs were recorded under dark-and light-adapted conditions from the corneal surface of nob2 mice, WT littermates and nob4 mice. ERG frequency spectra were calculated by fast Fourier transform (FFT). A FFT-based high-pass filter was used to derive OP waveforms. Under dark-adapted conditions, the dominant frequency of the OPs varied between 90 to 120 Hz in WT mice. In WT mice, OP frequency first increased with flash intensity and then decreased at the highest flash levels while overall OP amplitude increased monotonically with increasing flash intensity. In response to low stimulus flashes, reliable OPs were not obtained from nob2 mice. OPs were only seen at stimulus intensities at or above -1.8 log cd s/m(2), where they occurred at a lower frequency range (70-90 Hz) than for WT mice. When flash stimuli were superimposed against a steady rod-desensitizing adapting field, the amplitude and frequency of WT OPs increased with flash intensity above 0.4 log cd s/m(2). In comparison to WT results, cone-mediated OPs obtained from nob2 mice were smaller in amplitude, of lower frequency and had delayed implicit times. We compared the extent to which OPs and the b-wave were reduced in nob2 mice, by normalizing to the results obtained from WT mice. In comparison to the b-wave, the OPs were relatively spared, under both dark- and light-adapted conditions. In nob2 mice, rod- and cone-driven OPs are reduced in amplitude and occur at a lower frequency range. Since Ca(V)1.4 is expressed in both the inner and outer plexiform layers, these changes are likely to reflect reduced transmission from photoreceptors to bipolar cells as well as alterations in inner retinal function. That the OPs were better preserved than b-waves suggests that inner retinal pathways may be reorganized in response to the decreased bipolar cell response in nob2 mice.

Electroretinographic measures of vision in horseshoe crabs with uniform versus variegated carapaces.

The carapace characteristics of horseshoe crabs (Limulus polyphemus) have been correlated by others with male mating performance and it has also been suggested that such mating differences might depend on visual differences. The effect of carapace character on horseshoe crab vision was therefore noninvasively investigated using the electroretinogram (ERG) of the lateral compound eye as a measure. Male Limulus were dichotomized on three carapace dimensions: clear versus dark eyes, light versus dark carapaces, and presence versus absence of barnacles. All dichotomizations gave similar results: No crab was ERG-blind. Analyses based on ERG magnitude yielded trivial sensitivity differences between these groups. However, analyses based on ERG latency indicated that response speeds exhibited reliably different trends between groups: Although responses to dim flashes were similar for all animals, increasing flash intensity produced significantly greater changes in the latencies of the ERGs in uniform males than in variegated males. This interactive dependence of ERG speed differences on flash energy cannot be the result of greater light absorption by the variegated specimens' darkened corneas. The visual capacities of variegated specimens are therefore somewhat altered, but certainly not absent. Equalizing animal size between groups did not measurably affect these results, implying that age was not a factor. The previously reported correlation of an individual male's appearance with its performance in mating competitions is extended by these data to include its sensory functioning as well.

Photoreleased inositol 1,4,5-trisphosphate-induced response in single smooth muscle cells of rat portal vein.

1. The Ca2+ release in response to inositol 1,4,5-trisphosphate (InsP3) was studied in single patch-clamped smooth muscle cells of rat portal vein. InsP3 was photochemically produced from a caged InsP3 precursor included in the pipette solution. Changes in internal Ca2+ concentration ([Ca2+]i) were monitored by measuring Ca(2+)-activated K+ current. 2. Photoreleased InsP3 evoked a transient K+ current which was abolished when 10 mM EGTA or 5 mg ml-1 heparin was included in the pipette. The amplitude and time course of the K+ current responses depended on the light-flash intensity. The amplitude increased, and the latency and the time to peak decreased, with increasing flash intensity, suggesting that the amount of released Ca2+ varied as a function of the amount of InsP3 photoreleased. 3. The K+ current response to photolysis of caged InsP3 was abolished in the presence of 10 mM caffeine; conversely, caffeine was inefficient at inducing at K+ current when applied immediately after a light flash of maximal intensity. 4. The time course of the recovery of the K+ response evoked by a light flash of supramaximal intensity was similar to that obtained for the 10 mM caffeine-induced K+ current. The response recovered to 50% of control with an interval (t1/2) of about 10 s between pulses. The time course of the recovery of submaximal response to photoreleased InsP3 was considerably slower (t1/2 = 1 min), and did not correspond to that obtained for a response of similar amplitude evoked by 2 mM caffeine. 5. Responses to photoreleased InsP3 obtained after the cells were bathed for 3 min in Ca(2+)-free solution were compared with those obtained in 2 mM Ca2+ solution. Responses to light flashes of submaximal intensity were proportionally more inhibited than those evoked by supramaximal stimulations. 6. In portal vein smooth muscle cells, the InsP3-sensitive Ca2+ store seems also to be sensitive to caffeine. Our results suggest that the InsP3-induced Ca2+ release was modulated by regulatory mechanisms.

Two components of the receptor current are developed from distinct elementary signals in Limulus ventral nerve photoreceptor

Transient elementary currents, bumps, stimulated by short dim light flashes were measured in ventral nerve photoreceptors of Limulus. It is demonstrated that light activates two types of bumps, which form two distinct components of the receptor current at higher light intensities. The two bump types, which are both assumed to be activated by single absorbed photons, differ in current amplitude and kinetic parameters. The current amplitude of one bump type is smaller than 0.3 nA and that of the other type is in the usual current range of up to several nanoamperes. The average latency of small bumps measured from the short stimulus flash is shorter than that of the large bumps. The small bumps have slower activation kinetics than the large bumps. It is demonstrated that with increasing flash intensity the small bumps overlap first and form a macroscopic current, on top of which the large bumps are superimposed. Results indicate that a single absorbed photon selectively activates only one kind of the enzyme cascades evoking one bump type. We conclude that the active meta conformation of a rhodopsin molecule selectively binds a specific type of G-protein, which is involved in the stimulation of one of the transduction cascades. The two bump types, which are the elements of two macroscopic current components support the previous assumption that light activates different transduction mechanisms in Limulus photoreceptors.

Photoresponses and light adaptation of pineal photoreceptors in the trout

The photoresponses of 116 photoreceptors in the excised perfused pineal body of adult trout were intracellularly recorded. Brief flashes produced slower, but otherwise comparable, responses to retinal receptor cells. The absolute sensitivity, as well as the light-induced membrane conductance changes, were also similar to those of retinal cone cells in the same species. Steady illumination decreased the cell sensitivity to superimposed flashes, in accordance with the Weber-Fechner relation, indicating the presence of light adaptation. This conclusion is supported by the non-exponential nature of the amplitude-intensity relation and by the conspicuous shortening of the time-to-peak with increasing flash intensity. The time-dependent response decay typical of retinal photoreceptors was not found, however. The fact that, during constant illumination, the pineal photoresponses maintain the same amplitude set at the initial peak perhaps represents an original mechanism of light adaptation which allows these cells to maintain their voltage-dependent synthesis and secretion of indolamines as a constant relation with daily illumination.

Kinetic study of photo-induced electron transfer reactions sensitized by colloidal CdS

The kinetics of the recovery of the photoinduced transient bleaching of colloidal CdS in the presence of different electron acceptors are examined. In the presence of the zwitterionic viologen, N, N′-dipropyl-2,2′-bipyridinium disulphonate, excitation of colloidal CdS at different flash intensities generates a series of decay profiles which are superimposed when normalized. The shape of the decay curves are as predicted by a first-order activation-controlled model for a log-normal distribution of particles sizes. In contrast, the variation in flash intensity in the presence of a second viologen, N, N′-dipropyl-4,4′-bipyridinium sulphonate, generates normalized decay traces which broaden with increasing flash intensity. This behaviour is predicted by a zero-order diffusion-controlled model for a log-normal distribution of particle radii. The photoreduction of a number of other oxidants sensitized by colloidal CdS is examined and the shape of the decay kinetics interpreted via either the first- or zero-order kinetics models. The rate constants and activation energies derived using these models are consistent with the values expected for an activation- or diffusion-controlled reaction.

Response properties of cones from the retina of the tiger salamander.

1. Spectral sensitivity measurements using the suction electrode technique reveal three types of cone in the retina of the tiger salamander, showing maximum sensitivity at wavelengths 610 nm (red-sensitive cone), 444 nm (blue-sensitive cone) and below 400 nm (UV-sensitive cone). 2. The absolute sensitivities of red- and blue-sensitive cones to flashes of optimal wavelength are 0.022 and 0.33 pA photon-1 micron 2 respectively. 3. The time-to-peak of the dim flash response and the recovery of membrane current after a flash of any intensity are fastest in red-sensitive and slowest in blue-sensitive cones. 4. In blue- and UV-sensitive cones the flash response peaks progressively earlier as the flash strength is increased, as in rods. In red-sensitive cones, however, bright flash responses take longer to peak than dim flash responses. 5. In all three cone types, voltage clamping at -40 mV reduces the time-to-peak of the response to a bright flash, showing that the rising phase of the bright flash response is normally limited by the time constant of the cell. Under voltage clamp, all cones show a decrease in time-to-peak with increasing flash intensity. 6. Voltage clamping red-sensitive cones reveals two components of the rising phase of the response to a bright flash. Most of the current is rapidly suppressed by a bright flash, and represents the closure of light-sensitive channels. The residual current decays with a mean time constant of 20 ms, and is probably attributable to the decline of electrogenic Na(+)-Ca2+, K+ exchange. The amplitude of this exchange current suggests that the proportion of the dark current carried by calcium ions is greater in red-sensitive cones than in rods of the same species. 7. In UV-sensitive cones, a prominent oscillation of light-sensitive current is observed during the recovery from flashes of intermediate intensity. A similar, but slower and less prominent oscillation is usually seen in blue-sensitive cones. 8. When a red-sensitive cone is voltage clamped an oscillation similar to those in the other two cone types is revealed. An underswing of up to 2 pA is also observed after recovery from intermediate or bright flashes in the majority of red-sensitive cones, and voltage clamping increases the amplitude of this underswing.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of an electrical potential on the charge transfer kinetics of bacteriorhodopsin

The adsorption of bacteriorhodopsin(bR)-containing purple membranes (PM) to black lipid membranes (BLM) was used to study the charge translocation kinetics of bR upon flash excitation.The discharge of the PM-BLM system after charging upon illumination is found to proceed quite slowly (discharge time up to several minutes) but is considerably accelerated by addition of the protonophore FCCP.Therefore, the dependence of the proton transfer kinetics in bR on electrical potentials generated by preceding flashes of varying repetition rate and intensity was investigated. The kinetics are slowed down with increasing flash intensity as well as repetition rate. This effect is partly abolished by small amounts of FCCP.A new model is introduced which takes into account the instantaneous feedback of the electrical potential on the kinetics of the pump current. It explains the observed deviations from first-order kinetics and renders an approach with "distributed kinetics" unnecessary.

Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum.

1. Membrane currents initiated by intense, 20 microseconds flashes (photocurrents) were recorded from isolated salamander rods by combined extracellular suction electrodes and intracellular tight-seal electrodes either in current or voltage clamp mode. The magnitudes (mean +/- 2 S.E.M.) of the maximal photoresponses recorded by the suction and by the intracellular electrode respectively were 40 +/- 5 pA (n = 18) and 35 +/- 7 mV (n = 8) for current clamp at zero current; 43 +/- 9 pA and 66 +/- 13 (n = 11) pA for voltage clamp at the zero-current holding potential, -24 +/- 3 mV. 2. Photocurrents initiated by flashes isomerizing 0.1% or more of the outer segment's rhodopsin achieved a saturated velocity and were 95% complete in less than 50 ms. The effect of incrementing flash intensity above 0.1% isomerization can be described as a translation of the photocurrent along the time axis towards the origin. Within the interval 0-50 ms the latter two-thirds of the velocity-saturated photocurrent is well described as a single-exponential decay. The decay was much faster in voltage clamp (2.8 +/- 1.2 ms, n = 11) than in current clamp mode (17 +/- 5 ms, n = 17). 3. The initial third of the velocity-saturated photocurrent, occurring over the interval from the flash to the onset of exponential decay, followed about the same time course in current and voltage clamp. The time interval occupied by this initial 'latent' phase decreased with increasing flash intensity and attained an apparent minimum of about 7 ms in response to flashes isomerizing 10% or more of the rhodopsin at ca. 22 degrees C. 4. The hypothesis that the decay of outer segment light-sensitive membrane current is the same in current and voltage clamp was supported by an analysis of the difference between outer segment currents measured successively in the two recording modes. First, the tail of the difference current decayed exponentially with a time constant approximately equal to R x C, where R and C are independently estimated slope resistance and capacitance of the rod. Secondly, the integral of the difference current, when divided by outer segment capacitance, closely approximated the hyperpolarizing light response measured under current clamp. Thus, displacement current accounted for the difference between photocurrents measured in current and voltage clamp.(ABSTRACT TRUNCATED AT 400 WORDS)

Antenna and reaction-center processes upon picosecond-flash excitation of membranes of the green photosynthetic bacterium Chloroflexus aurantiacus

The formation and decay of antenna-excited states and the primary charge separation in membranes of the green photosynthetic bacterium Chloroflexus aurantiacus were studied by means of picosecond absorbance difference spectroscopy. After chemical oxidation of the primary electron donor, a 35 ps excitation pulse at 532 nm produced singlet- and triplet-excited states of carotenoid and of bacteriochlorophyll a. Excitation of bacteriochlorophyll a caused a bleaching of its Q y absorption band and induced a blue shift of several neighboring bacteriochlorophyll molecules. The singlet-excited state decayed biphasically with lifetimes of about 200 ps and 1.2 ns. A decrease in the lifetime at increasing flash intensity was attributed to singlet-singlet annihilation. In the presence of active reaction centers also the primary-charge separation and secondary electron transfer were observed. The charge separation consisted of the transfer of an electron from the primary donor, P-865, to the primary-acceptor complex of bacteriopheophytin a and bacteriochlorophyll a. Electron transfer to a secondary acceptor occurred with a time constant of 400 ± 50 ps, which is about 30% longer than had been observed with isolated reaction centers (Kirmaier, C., Holten, D., Mancino, L.J. and Blankenship, R.E. (1984) Biochim. Biophys. Acta 765, 138–146). When this secondary acceptor was prereduced chemically, the lifetime of the primary radical pair increased to 10 ns or more.

The initial response of Limulus ventral photoreceptors to bright flashes. Released calcium as a synergist to excitation.

The leading edge of the response of Limulus ventral photoreceptors to brief flashes was investigated using a voltage clamp. The leading edge of responses increases linearly with flash intensity when dim flashes produce less than one photoisomerization per square micron of cell surface. Brighter flashes accelerate the initial portion of the response, resulting in a fourth-power relationship between the magnitude of the response at brief times after the flash and the flash intensity. The onset of this nonlinearity with increasing flash intensity is determined by the local density of photoisomerizations within the receptor. Responses to bright 10-15-mum-diam spots therefore rise faster than responses to diffuse flashes producing the same number of photoisomerizations within the receptor. Background illumination shortens the response latency and suppresses the initial nonlinearity. These phenomena can be explained by a model of transduction in which light activates two parallel cascades of reactions. Particles released by the first of these cascades open ionic channels, while the second produces an agent that accelerates the rate of production of particles by the first. Injection of the calcium buffer EGTA slows the initial portion of the response to bright flashes and suppresses its nonlinearity, which suggests that the accelerating agent released by the second cascade is calcium.

EFFECT OF PHOTOSELECTION UPON SATURATION AND THE DICHROIC RATIO IN FLASH EXPERIMENTS UPON EFFECTIVELY IMMOBILIZED SYSTEMS

Abstract— Flash spectroscopy of photochemical or photobiological systems, such as bacteriorhodopsin in purple membrane, for which the chromophore transition dipole moment does not undergo complete reorientation during the time of the flash, is considered as a function of light intensity. Due to photoselection, saturation proceeds very slowly with increasing flash intensity and the linear dichroic ratio decays rapidly from a maximum of three at zero flash intensity. Simple formulae are derived to describe these effects under stringent assumptions. Calculations are also performed which relax the assumptions by taking into account (i) non‐zero optical density, (ii) thermal decay of the photoproduct during the flash and (iii) non‐zero angle y between the initial chromophore and its photoproduct. Agreement with experiments on bacteriorhodopsin in purple membranes is excellent.

Two definitions of persistence in visual perception.

It is well known that the perception of a visual stimulus does not terminate with the offset of that stimulus, but decays with time. Although a variety of specific methods have been developed to study this phenomenon, these methods have not always yielded consistent results. We believe that some of the discrepancies in the literature on visual persistence have arisen because different researchers have based their estimates of persistence on qualitatively different features of the visual residual. Efron (1970) asked subjects to temporally align the onset of a click with the offset of a brief flash. Persistence, defined as the interval between flash offset and onset of the indexing click, was found to decrease with increasing flash intensity. Bowen, Pola, and Matin (1974) asked subjects to adjust the temporal placement of a probe flash so that it was coincident with the apparent offset of a spatially separated test flash. Persistence was defined as the interval between test flash offset and onset of the indexing flash. Again, measured persistence decreased with increasing test stimulus intensity. The same result has been reported by Hansteen (1971) using a procedure resembling that of Bowen et al. In a somewhat different approach, Haber and Standing (1969) presented a repetitive cycle in which a stimulus was followed by a blank interval. Subjects were asked to adjust the duration of the blank interval between successive stimulus presentations so as to produce a discontinuity. Once again, persistence, defined by the duration of the blank interval between successive stimuli, declined with increases in the intensity of the test stimulus. Other researchers, however, using different procedures, have reported that stimulus intensity has the opposite effect on persistence. In one of a series of experiments, Sakitt (1976) asked observers to temporally align the onset of a click with the disappearance of the residual (icon) of a briefly exposed letter array. Her measure of persistence, the temporal inter-

Direct observation of a light-induced electric field in chloroplasts

1. We report the first direct measurement of an electric field generated by the photoevents in photosynthesis. 2. Illumination of structurally intact and photochemically active spinach chloroplasts with a short non-saturating flash generates a transient dipole field. 3. Photosystem I and Photosystem II contribute equally to the steady-state signals in a sequence of flashes. 4. The rise time of the transient is extremely rapid, reflecting early photochemical events. In the time range studied, the decay time of the field is inversely proportional to the conductance of the suspending medium and independent of photosynthetic ion or electron transport. 5. The polarity of the signal is determined by the intensity gradient of the light field; the electrode nearest the light source becomes negative. 6. With increasing flash intensity, the amplitude of the transient goes through a maximum which implies that the signal results from two oppositely directed polarizations. 7. We conclude that the electric field arises from the paired membranes of the chloroplast which contain the photosystems. The phototraps are arranged so that during the photoacts, charges move perpendicular to the plane of each membrane. The donors of both Photosystems I and II are located on the “interior” of the membrane systems and the acceptors are located “outside”. During illumination electrons move to the outside, polarizing the two membranes in opposite direction.

An allosteric model of hemoglobin: I, kinetics

The allosteric model of Monod, Wyman & Changeux (1965; the Monod model) has been used to analyze the kinetics of ligand binding to mammalian hemoglobins. Combination velocity constants and dissociation velocity constants for the T and R forms were evaluated from the early measurements by Roughton & Gibson of the oxygen and carbon monoxide binding to sheep hemoglobin at pH 9.1 and more recent measurements of oxygen binding to human hemoglobin at pH 7. The difference in ligand affinities between the T and R forms, which is a factor of several hundred, is taken up almost equally by the combination and dissociation rates. The allosteric model is shown to be particularly suitable for explaining the flash photodissociation of carboxyhemoglobin. The monotonic decrease of the fraction of quickly reacting form with increasing flash intensity shown by Antonini, Chiancone & Brunori (1967) and the flow-flash experiments of Gibson & Parkhurst (1968) are both in excellent agreement with the calculations on the Monod model, with no adjustable parameters except for the fit to the equilibrium curves. In flow experiments, both the forward progess curves and the reverse dissociation curves after mixing with dithionite are very well described by the Monod model. Finally, Gibson's recent flow experiments on human hemoglobin at pH 7 have been shown to be consistent with the parameters of the Monod model which can describe the other kinetic experiments. From all this, we conclude that the kinetic experiments discussed can be fitted by the simple Monod allosteric model to well within the range of experimental errors, and that the kinetic data now available are not inconsistent with this model.