Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

The galactoside-specific Viscum album L. agglutinin (VAA) is a potent biohazard akin to ricin and a mitogen for immune and tumor cells. These activities depend on cell surface binding to glycans. It is an open question whether the process of ligand binding alters the lectin's shape. Small angle neutron scattering (SANS) experiments revealed that the carbohydrate ligand lactose induced a decrease of the radius of gyration of dimeric VAA from 54.5 +/- 1 to 49.5 +/- 1 A in water. Apparently, VAA in aqueous solution and at the concentrations tested at 3.6 mg/ml and above adopts a compacted structure as response to ligand binding. In contrast to the behavior in aqueous solution, lactose binding in DMSO resulted in an increase of the lectin's radius of gyration from 49 +/- 1 to 55.5 +/- 1 A. Because shape changes may be reflected in the thermostability of the protein, this parameter was examined by activity assays of protein exposed to 60 degrees C and 70 degrees C and by differential scanning calorimetry (DSC). In line with the lactose-induced conformational alterations revealed by the SANS experiments, lactose presence enhanced the thermostability of VAA in water. Thus, binding of the carbohydrate ligand in solution can entail changes in shape and thermostability in the case of the tested plant lectin.     

Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers

To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha v beta 3 for this ligand, we used laser tweezers to measure the rupture force between single alpha v beta 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 microM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, approximately 10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha v beta 3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet alpha v beta 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha v beta 3-binding sites for this ligand.     

Low spring constant regulates P-selectin-PSGL-1 bond rupture

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r_f (≥10² pN/s), defined as the product of spring constant k and retract velocity v, how the low r_f (<10² pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r_f ≤ 20 pN/s with low k (∼10⁻³-10⁻² pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r_f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r_f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 × 10⁻³ pN/nm, indicating that the bond dissociation at low r_f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r_f or k.     

Protein adhesion force dynamics and single adhesion events.

Using the manipulation force microscope, a novel atomic force microscope, the adhesion forces of bovine serum albumin, myoglobin, ferritin, and lysozyme proteins to glass and polystyrene substrates were characterized by following the force necessary to displace an adsorbed protein-covered microsphere over several orders of magnitude in time. This force was consistent with a power law with exponent a = 0.37 +/- 0.03 on polystyrene, indicating that there is no typical time scale for adhesion on this substrate. On glass, the rate of adhesion depended strongly on protein charge. Forces corresponding to single protein adhesion events were identified. The typical rupture force of a single lysozyme, ferritin, bovine serum albumin, and myoglobin protein adhering to glass was estimated to be 90 +/- 10 pN, 115 +/- 13 pN, 277 +/- 44 pN, and 277 +/- 44 pN, respectively, using a model of the experimental system. These forces, as well as the force amplitudes on hydrophobic polystyrene, correlate with protein stiffness.     

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies

There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies.     

Differences in cardiac microcirculatory wave patterns between the proximal left mainstem and proximal right coronary artery

Despite having almost identical origins and similar perfusion pressures, the flow-velocity waveforms in the left and right coronary arteries are strikingly different. We hypothesized that pressure differences originating from the distal (microcirculatory) bed would account for the differences in the flow-velocity waveform. We used wave intensity analysis to separate and quantify proximal- and distal-originating pressures to study the differences in velocity waveforms. In 20 subjects with unobstructed coronary arteries, sensor-tipped intra-arterial wires were used to measure simultaneous pressure and Doppler velocity in the proximal left main stem (LMS) and proximal right coronary artery (RCA). Proximal- and distal-originating waves were separated using wave intensity analysis, and differences in waves were examined in relation to structural and anatomic differences between the two arteries. Diastolic flow velocity was lower in the RCA than in the LMS (35.1 +/- 21.4 vs. 56.4 +/- 32.5 cm/s, P < 0.002), and, consequently, the diastolic-to-systolic ratio of peak flow velocity in the RCA was significantly less than in the LMS (1.00 +/- 0.32 vs. 1.79 +/- 0.48, P < 0.001). This was due to a lower distal-originating suction wave (8.2 +/- 6.6 x 10(3) vs. 16.0 +/- 12.2 x 10(3) W.m(-2).s(-1), P < 0.01). The suction wave in the LMS correlated positively with left ventricular pressure (r = 0.6, P < 0.01) and in the RCA with estimated right ventricular systolic pressure (r = 0.7, P = 0.05) but not with the respective diameter in these arteries. In contrast to the LMS, where coronary flow velocity was predominantly diastolic, in the proximal RCA coronary flow velocity was similar in systole and diastole. This difference was due to a smaller distal-originating suction wave in the RCA, which can be explained by differences in elastance and pressure generated between right and left ventricles.     

Ligand binding subsequent to CO photolysis of methionine-modified cytochrome c

In this report the kinetics of CO recombination to ferrocytochrome c in which Met80 has been oxidized to a sulfoxide are examined. Transient optical difference spectra suggest that the species formed immediately after photolysis contains a five-coordinate high spin heme. Single wavelength transient absorption data show triphasic kinetics with rate constants of (2.1+/-0.08)x10(4), (2.0+/-0.01)x10(3), and 57+/-0.01 s(-1). The data suggest a model for ligand recombination in which the methionine sulfoxide and CO compete for binding to the five-coordinate heme with rate constants of (2.1+/-0.08)x10(4) and (2.0+/-0.01)x10(3) s(-1), respectively. Carbon monoxide then binds to the population of cytochrome c containing the methionine sulfoxide with a rate constant of 57 s(-1). In addition, the slower than expected rate of methionine sulfoxide recombination (much smaller rate constant than expected for a ligand restricted to the distal heme pocket) is attributed to hydrogen bonding between the unbound methionine sulfoxide and Tyr(68).     

Force spectroscopy of the double-tethered concanavalin-A mannose bond

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.     

Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy

The regulated ability of integrin alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of nonspecific protein-protein binding versus specific alphaIIbbeta3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alphaIIbbeta3 and fibrinogen, covalently attached to underlying surfaces, ranged from approximately 20 to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20-60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80-90 pN. The interactions corresponding to these rupture force regimes differed in their susceptibility to alphaIIbbeta3 antagonists or Mn2+, an alphaIIbbeta3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >3.5-fold but did not affect the shape of the rupture force distribution, indicating that the measurements represent single-molecule binding. The yield strength of alphaIIbbeta3-fibrinogen interactions was independent of the loading rate (160-16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of alphaIIbbeta3 and fibrinogen revealed at the single-molecule level.     

Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Double-tether extraction from human umbilical vein and dermal microvascular endothelial cells

Multiple tethers are very likely extracted when leukocytes roll on the endothelium under high shear stress. Endothelial cells have been predicted to contribute more significantly to simultaneous tethers and thus to the overall rolling stabilization. We therefore extracted and quantified double tethers from endothelial cells with the micropipette aspiration technique. We show that the constitutive parameters (threshold force (F0) and effective viscosity (etaeff)) for double-tether extraction are twice those for single-tether extraction and are remarkably similar for human neonatal (F0=105+/-5 pN; etaeff=1.0+/-0.1 pN.s/microm) and adult (F0=118+/-13 pN; etaeff=1.3+/-0.2 pN.s/microm) dermal microvascular, and human umbilical vein (F0=99+/-3 pN; etaeff=1.0+/-0.1 pN.s/microm) endothelial cells. Additionally, these parameters are also independent of surface receptor type, cytokine stimulation, and attachment state of the endothelial cell. We also introduce a novel correlation between the cell-substrate contact stress and gap width, with which we can predict the apparent cell-substrate separation range to be 0.01-0.1 microm during leukocyte rolling. With a biomechanical model of leukocyte rolling, we calculate the force history on the receptor-ligand bond during tether extraction and predict maximum stabilization for the double simultaneous tether extraction case.     

A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds

A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-microm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewis(x), was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), r(f). From the distribution of forces at failure, the average force was determined and plotted as a function of ln r(f). The slope and intercept of the plot yield the unstressed reverse reaction rate, k(r)(o), and a parameter that describes the force dependence of reverse reaction rates, r(o). The ligand was titrated so adhesion occurred in approximately 30% of tests, implying that >80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are r(o) = 0.048 and 0.016 nm and k(r)(o) = 0.72 and 2.2 s(-1) for loading rates in the ranges 200-1000 and 1000-5000 pN s(-1), respectively. Levenberg-Marquardt fitting across all values of r(f) gives r(o) = 0.034 nm and k(r)(o) = 0.82 s(-1). The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations.     

The Molecular Mechanics of P- and L-Selectin Lectin Domains Binding to PSGL-1

A laser trap was used to compare the load-dependent binding kinetics between truncated P- and L-selectin to their natural ligand, P-selectin glycoprotein ligand-1 (PSGL-1) over the predicted physiological range of loading rates. Human PSGL-1 was covalently coupled to polystyrene beads. Chimeric selectins were adsorbed to nitrocellulose-coated glass beads on a coverslip. A PSGL-1 bead was held in a laser trap and touched to a vertical surface of the glass bead, allowing a bond to form between selectin and ligand. The surface was moved away from the microsphere, applying load at a constant rate until bond rupture. Rupture force for both selectins increased with loading rate, but significant differences in rupture force between P- and L-selectin were observed only above 460 pN/s. These data are best represented as two energy barriers to unbinding, with the transition from the low to high loading rate regime at 260–290 pN/s. The data also allow the first estimate of a two-dimensional specific on-rate for binding of these two selectins to their natural ligand (1.7 μm²/s). These data suggest that P- and L-selectin lectin domains have very similar kinetics under physiological conditions.     

Single molecule characterization of P-selectin/ligand binding

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.     

Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. II. Protein-protein bond.

In an extension of the previous paper, we describe the force dependence of break-up of doublets of latex spheres cross-linked by protein G-IgG bonds via the Fc region of the antibody. The receptor, the monoclonal Bear-1 antibody, was either covalently linked to 4.75-microns aldehyde/sulfate (A/S) latex spheres in a one-step reaction, or physically adsorbed to the 4.63-microns carboxyl-modified latex spheres used in Part I of this paper. The spheres were suspended in 19% buffered Dextran 40 containing the ligand, the bivalent recombinant protein G (Gamma-Bind G), and observed in the counter-rotating cone and plate Rheoscope. Break-up of doublets, tracked individually under the microscope, as well as in populations of 50-150 particles, was studied over a range of normal force from 20 to 260 pN. In individual particle studies, the fraction of doublets of spheres with covalently linked IgG breaking up in the first 10 rotations, increased from 16% in the low-force to 63% in the high-force range. In population studies, the fraction broken up increased with duration and magnitude of the applied force, and decreased with increasing ligand concentration. Moreover, doublets of physically adsorbed IgG spheres required significantly lower force than doublets of covalently linked IgG spheres for the same degree of break-up, possibly because of surface detachment of IgG molecules rather than rupture of receptor-ligand bonds. Computer simulation, using the Bell stochastic model of break-up and a Poisson distribution for the number of bonds, described in Part I, showed that the parameters of the protein-protein bond differed significantly from those of the carbohydrate-protein bond studied in Part I of this paper, the former being much more responsive to force than the latter.     

Measurement of the force produced by an intact bull sperm flagellum in isometric arrest and estimation of the dynein stall force

The force generated by a detergent-extracted reactivated bull sperm flagellum during an isometric stall was measured with a force-calibrated glass microprobe. The average isometric stall force from 48 individual measurements was 2.5 +/- 0.7 x 10(-5) dyne (2.5 +/- 0.7 x 10(-10) N). The force measurements were obtained by positioning a calibrated microprobe in the beat path of sperm cells that were stuck by their heads to a glass microscope slide. The average position of the contact point of the flagellum with the probe was 15 microm from the head-tail junction. This average lever arm length multiplied by the measured force yields an estimate of the active bending moment (torque) of 3.9 x 10(-8) dyne x cm (3.9 x 10(-15) N x m). The force was sustained and was for the most part uniform, despite the fact that the flagellum beyond the point of contact with the probe usually continued beating. It appears that the dynein motors in the basal portion of the flagellum continue to pull in an isometric stall for as long as the motion of the flagellum is blocked. If dynein motors in the flagellum distal to the contact point with the probe were contributing force to the displacement of the probe, then the flagellar segment immediately past the point of contact would have to show a net curvature in the direction of the probe displacement. No such curvature bias was observed in the R-bend arrests, and only a small positive curvature bias was measured in the P-bend arrests. Our analysis of the data suggests that more than 90% of the sustained force component is generated by the part of the flagellum between the probe and the flagellar base. Based on this premise, the isometric stall force per dynein head is estimated to be 5.0 x 10(-7) dyne (5 pN). This equals approximately 1.0 x 10(-6) dyne (10 pN) per intact dynein arm. These values are close to the isometric stall force of isolated dynein. This suggests that all of the dynein heads between the base and the probe, on the active side of the axoneme, are contributing to the force exerted against the probe.     

Responses of Rubisco and Rubisco activase in cucumber seedlings to low temperature and weak light

Using 'Jinyou 3' cucumber seedlings as test materials, this paper studied their photosynthetic rate (P(n)), Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (RCA) activities, and gene expression of Rubisco and RCA under optimal temperature and weak light (WL: 25 degrees C/18 degrees C, 100 micromol x m(-2) x s(-1)), suboptimal temperature and weak light (ST+WL: 18 degrees C/12 degrees C, 100 micromol x m(-2) x s(-1)), and low temperature and weak light (LT+WL: 10 degress C/5 degrees C, 100 micromol x m(-2) x s(-1)). Comparing with the control (25 degrees C/18 degrees C, 400 micromol x m(-2) x s(-1)), treatments WL, ST+WL, and LT+WL all led to a remarkable decrease in leaf area and dry matter mass. At initial stage, the P(n), Rubisco activity, rbcL and rbcS expression, RCA activity, and CsRCA expression in the three treatments declined by a big margin; 5-7 days later, these parameters tended to be less changed in treatment WL, ascended slowly in treatment ST+WL, and decreased continuously in treatment LT+WL. These results suggested that the photosynthetic apparatus of test cucumber seedlings could gradually adapt to weak light or suboptimal temperature and weak light. The Rubisco and RCA activities and the gene expression of Rubisco and RCA showed the similar responses to low temperature and weak light as the P(n), suggesting that the decline in Rubisco and RCA activities and gene expression in cucumber seedlings under low temperature and weak light could be the important reason leading to the decrease of P(n).     

Free radical-induced redox chemistry of platinum-containing anti-cancer drugs

Arrhenius parameters for the reactions of oxidizing hydroxyl radicals and reducing hydrated electrons with cisplatin, transplatin and carboplatin in aqueous solution have been determined using pulsed electron radiolysis and absorption spectroscopy techniques. Under physiological pH and chloride concentration conditions, hydroxyl radical reaction rate constants of (9.99 +/- 0.20) x 10(9), (8.38 +/- 0.55) x 10(9), and (6.03 +/- 0.08) x 10(9) M(-1) s(-1) at 24.0, 20.7 and 24.0 degrees C, respectively, with corresponding activation energies of 12.79 +/- 0.57, 13.88 +/- 1.14, and 14.35 +/- 0.56 kJ mol(-1) for these three reactions, were determined. These oxidations of cisplatin and transplatin to form a Pt(III) transient are significantly faster than reported previously at room temperature. The lower rate constant for carboplatin is consistent with hydroxyl radical reaction partitioning between reaction at the platinum center and the cyclobutanedicarboxylate ligand. The equivalent reductive hydrated electron reaction rate constants measured were (1.99 +/- 0.04) x 10(10) (24.0 degrees C), (1.77 +/- 0.08) x 10(10) (22.0 degrees C), and (8.92 +/- 0.06) x 10(9) M(-1) s(-1) (24.0 degrees C), with corresponding activation energies of 15.75 +/- 1.00, 19.74 +/- 1.82, and 19.99 +/- 0.34 kJ mol(-1). Again, the values determined for cisplatin and transplatin are faster than reported; however, all three values are consistent with direct reduction of the platinum center to form a Pt(I) moiety.