Water proton relaxation rate enhancements and association constants for Mn(II) to serum proteins determined by NMR T1 measurements

The water proton relaxation rate enhancement of Mn(II) bound to bovine serum albumin (BSA) and the association constant for manganese to BSA have already been determined, but such determinations have not been done for human serum albumin (HSA) and other human serum proteins and also for human serum. In this work, NMR T1 values in aqueous solutions of serum proteins and serum were measured versus increasing concentration of Mn(II). Proton relaxation rate enhancements (epsilon*) caused by different manganese concentrations were determined for each solution and 1/epsilon* was fitted against concentrations of Mn(II). Proton relaxation rate enhancements (epsilonb) of Mn(II) bound to albumin, gamma-globulin, (alpha+beta)-globulins and serum were found to be 13.69, 3.09, 8.62, and 10.87, respectively. Free and bound manganese fractions, resulted from each addition of Mn(II) to the sample, were determined by using corresponding (epsilon*) and the epsilonb values. Association constants for Mn(II) to HSA and gamma-globulin were calculated as 1.84 x 10(4) M(-1) and 2.35 x 10(4) M(-1), respectively. Present data suggest that the proton relaxation rate enhancement of Mn(II) in serum is caused by Mn(II) bound to various serum constituents. Data also suggest that association constants for Mn(II) to gamma-globulin are nearly the same as that to HSA.     

Site I on human albumin: differences in the binding of (R)- and (S)-warfarin.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.     

Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin

A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5.     

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.     

Influence of paramagnetic ions bound to human serum albumin on water 1HNMR relaxation times

The extent to which various paramagnetic ions (Cu2+, Mn2+ and Gd3+) free and bound to human serum albumin alter the water proton relaxation times at two frequencies has been investigated. NMR relaxation parameters, T1 and T2, were measured at 5 and 10 MHz using a saturation recovery (90 degrees-tau-90 degrees) and a spin-echo (90 degrees-tau-180 degrees) sequence respectively. We found that all three ions enhance their effectiveness in inducing water proton magnetic relaxation when they are bound to human serum albumin and that Gd3+ is the most effective in pure water and Mn2+ in the presence of the protein. Cu2+ has a smaller effect, but it presents an interesting behaviour correlated with the existence of two different binding sites, which is also confirmed by electronic paramagnetic resonance spectra. The results indicate the potential usefulness of large molecular paramagnetic complexes as contrast agents in NMR Imaging.     

Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin, and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which had been demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 X 10(-12) M [Agarwal, R. P., Spector, T., & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, 359-367], (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 microM for inhibition by (S)-deoxycoformycin, 43 microM for 8-ketodeoxycoformycin, and 16 microM for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1.3 X 10(7]-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analogue of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism, and chemical criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Warfarin metabolites: Stereochemical aspects of protein binding and displacement by phenylbutazone

The in vitro human serum albumin binding characteristics of the enantiomers of the major metabolites of warfarin [6-hydroxywarfarin (6-HW), 7-hydroxywarfarin (7-HW), (S)-warfarin alcohols [(S,S)- and (S,R)-WA], and (R,S)-warfarin alcohol [(R,S)-WA]] have been studied, using a stereospecific HPLC assay. Warfarin metabolites are less bound both within plasma and a 40 g/liter solution of human serum albumin than the enantiomers of warfarin. The reduced warfarin metabolites have a lower fraction unbound [1.33% for (S,R)-WA, 2.09% for (S,S)-WA, and 1.04% for (R,S)-WA] than hydroxylated metabolites [3.24% for (R)-6-HW, 4.26% (S)-6-HW, 4.49% for (R)-7-HW and 4.27% for (S)-7-HW] to HSA. Phenylbutazone produced a concentration-dependent increase in the unbound fraction of all metabolites. It was possible to predict the unbound fraction of warfarin metabolites based on the unbound fraction of warfarin enantiomers. © 1993 Wiley-Liss, Inc.     

Selective antibodies to methadone enantiomers: synthesis of (R)- and (R,S)-methadone conjugates and determination by an immunoenzymatic method in human serum

Selective antibodies to (R)-methadone (Mtd) and to its racemate were produced in rabbits by immunization with conjugates of (R)- or (R,S)-hemisuccinyl-methadol-bovine serum albumin, respectively. A hapten was first prepared by reduction of (R)- or (R,S)-Mtd with sodium borohydride, followed by esterification with succinic anhydride. The conjugation of hapten with albumin was achieved by the mixed anhydride method. After immunization of rabbits, the titers and specificity of each antibody were determined by ELISA. The antibodies obtained were tested with (R)-, (S)-, (R,S)-Mtd, its major metabolite (EDDP), and some drugs of abuse (morphine, codeine, cocaine). The sensitivities of antibodies to (R)- and (R,S)-Mtd were about 1 and 2 ng/ml, respectively. Selective (R)-antibodies recognized (R)-Mtd about 40 times more avidly than the (S)-isomer, while an antiserum against (R,S)-Mtd recognized (R)- and (S)-isomers to about the same degree. Both selective antibodies showed little interference (about 0.5%) with EDDP metabolite and no crossreactivity with morphine, codeine, and cocaine. These two selective antibodies were used to develop an immunoenzymatic method (ELISA) for the determination of (R)- and (R,S)-Mtd in serum samples of patients under maintenance treatment for narcotic addiction.     

Stereoselective protein binding of ketoprofen: Effect of albumin concentration and of the biological system

Equilibrium dialysis was used to study in vitro the enantioselective binding of R, S, and racemic ketoprofen at physiological pH and temperature in human serum albumin (HSA) (1, 20, and 40 g/liter) and in plasma. The binding of enantiomers in a racemic mixture was studied to see the effect of each isomer on the other's interaction with the protein. The free fractions were determined by high-performance liquid chromatography. The binding of ketoprofen enantiomers to albumin was enantioselective, depending on both drug and protein concentrations. Enantioselectivity was observed in plasma too but was the opposite of that in HSA at 40 g/liter. The percentage of each isomer unbound was higher in the racemic mixture than with the isomer alone. The displacement of probes specific for HSA sites I and II, studied by spectrofluorimetry, suggests that all three preparations of ketoprofen are bound mainly to site I and secondarily to site II. © 1993 Wiley-Liss, Inc.     

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.     

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.     

Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies.

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.     

Inversion of (R)- to (S)-ketoprofen in eight animal species

The (R)-enantiomer of the NSAID ketoprofen was administered orally at 20 mg/kg to a series of 8 animal species. In all species, a highly significant degree of inversion occurred after 1 h which varied from 27% (gerbil) to 73% (dog) and persisted or increased in plasma samples obtained 3 h after drug administration. Although the (R)-enantiomer was inactive as an inhibitor of cyclooxygenase, the analgesic effects of that isomer was almost the same as the (S)-isomer in animal analgesic assays, following oral administration of the drugs to mice and rats. Taken together, the present results suggest that (R)-ketoprofen administered alone functioned primarily as a prodrug for (S)-ketoprofen under the experimental conditions of this study. © 1995 Wiley-Liss, Inc.     

Epimerization of moxalactam by albumin and simulation of in vivo epimerization by a physiologically based pharmacokinetic model

We investigated the mechanism of epimerization (R to S or S to R) of moxalactam in serum of rats, dogs, and humans. The epimerization of moxalactam occurred in the serum of these animals, but not in the serum filtrate. The albumin fraction of human serum purified by gel filtration catalysed the epimerization of moxalactam at an identical rate to serum, but other fractions (i.e., lipoproteins and globulins) showed slower epimerization. alpha 1-acid glycoprotein, which was eluted in the same fraction with albumin by G-200 gel filtration, did not epimerize moxalactam. The presence of 2 mM warfarin decreased the binding of R- and S-moxalactam and decreased the epimerization of moxalactam in human serum. These results demonstrate moxalactam was epimerized on the warfarin binding site on albumin in serum. Additionally, a physiologically based pharmacokinetic model shows that the epimerization of moxalactam after administration in dogs is simulated by the epimerization in serum.     

NMR study of the effect of sugar-phosphate backbone ethylation on the stability and conformation of DNA double helix

Temperature variation studies of the imido proton NMR and 31P NMR resonances of the self-associated d(C-C-A-A-G-A-T-T-G-G) and d[C-C-A-A-G-p(Et)-A-T-T-G-G] duplexes (both the R and S diastereoisomers) and the heteroduplexes formed with their complementary strand d(C-C-A-A-T-C-T-T-G-G) were carried out in aqueous solution. Results demonstrate that phosphate backbone ethylation did not disrupt the interstrand hydrogen bonding involved in double-helix formation but perturbed the helix. The S isomer perturbed the duplex more than the R isomer. The line broadening patterns and faster fraying motion in the alkylated duplexes compared to those in the nonalkylated duplexes indicate that the perturbation introduced in the middle propagates along the backbone to the end of the duplex.     

Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.     

The chirality selectivity in the uptake of platinum (II) complexes with 1,2-cyclohexanediamine isomers as carrier ligand by human erythrocytes

The uptake kinetics of cisplatin analogs of 1,2-cyclohexanediamine(dach) isomers with various leaving groups, by human erythrocytes in plasma isotonic buffer, were studied. The experimental results showed that the uptake rate constants (k values) decrease with the change of leaving group in the sequence: chloride (Cl) > squaric acid (SA) > oxalate (OX) > demethylcantharic acid (DA), with the same dach isomer as carrier group. It is noteworthy that for the platinum (II) complexes with the same leaving group, the k values always reduce as: 1R, 2R-dach > 1R, 2S-dach > 1S, 2S-dach. This result reflects the chirality selectivity. No differences in reactivity to protein thiols and effects on membrane permeability were found for the R,R-, R,S-, S,S-isomeric complexes. It is proposed that the chirality selectivity in uptake is due to the recognition of the chirality of the platinum complexes by the erythrocyte membrane. The interactions between the chiral platinum complexes and the head groups of the membrane phospholipid molecules are probably involved.     

Isomerization of (R)- and (S)-glutathiolactaldehydes by glyoxalase I: the case for dichotomous stereochemical behavior in a single active site.

The ability of glyoxalase I to isomerize both diastereomeric thiohemiacetals formed between glutathione and alpha-ketoaldehydes has been probed with stereochemically "locked" substrate analogues. Both (R)- and (S)-glutathiolactaldehyde (5 and 5') were unambiguously synthesized by employing the Sharpless epoxidation procedure as a key step. In the presence of human erythrocyte glyoxalase I, high-field 1H NMR analysis reveals that the R and S isomers (approximately 20 mM) are both converted to glutathiohydroxyacetone at rates of 0.8 and 0.4 s-1, respectively. This reaction is characterized by a nonstereospecific proton abstraction followed by a partially shielded proton transfer to the si face of the cis-enediol intermediate. Glyoxalase I catalyzes the exchange of the pro-S proton of glutathiohydroxyacetone with solvent deuterium. Glutathiohydroxyacetone was found to be a good competitive inhibitor of the normal glyoxalase I reaction (KI = 1.46 mM), suggesting that the slow processing rate of these compounds with respect to the normal thiohemiacetals is not due to poor binding. The results are consistent with a nonstereospecific proton abstraction and a stereospecific reprotonation at contiguous substrate carbons.     

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7)-10(-4) M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S),12(R),15(S),5(Z),8(Z),13(E)-THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R),12(R),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.     

Binding and photochemistry of enantiomeric 2-(3-benzoylphenyl)propionic acid (ketoprofen) in the human serum albumin environment.

Global analysis of circular dichroism multiwavelength data and time resolved fluorescence was applied to investigate the interaction of R(-)- and S(+)-ketoprofen (KP) with human serum albumin (HSA) in buffer solution at neutral pH. The most stable drug:protein adducts of 1 : 1 and 2 : 1 stoichiometry were characterized as regards the stability constants and the absolute circular dichroism spectra. The spectra of the diastereomeric 1 : 1 conjugates are negative with minima at ca. 350 nm for R(-)-KP and 330 nm for S(+)-KP, those of the 2 : 1 complexes are both negative with minimum at 340 nm and quite similar in shape to each other, thereby showing that the protein loses chiral recognition capability upon multiple binding. HSA intrinsic time resolved fluorescence data obtained exciting at 295 nm point to Trp 214 being located in the secondary binding site for both KP enantiomers. The photodegradation of the S(+)- and R(-)-KP:HSA complexes was studied by steady state photolysis using lambda(irr) > 320 nm. No decrease of the photodegradation quantum yields was observed in 1 : 1 complexes. An induction time for the photodegradation course in 2 : 1 complexes was observed. Transient absorption spectroscopy at lambda(exc) = 355 nm showed that triplet KP species were formed with stereo-differentiated lifetimes and high quantum yields (0.7-0.9). Secondary transients were consistent with the occurrence of photodecarboxylation and/or photoreduction within the protein matrix.