The photolysis of HbO2 and HbCO has been studied by measuring transient absorption spectra in the Soret region after excitation with picosecond pulses at 530 nm. Dissociation occurred promptly in both cases, followed (for HbO2) by geminate recombination of ca. 40% of the photodissociated O2 with a lifetime of 200 ± 70 psec (25°C). No to 300 psec. The initial MbCO photoproduct signal at 440 nm was reported to undergo a 15% decay (lifetime r = 125 + 50 psec), but for MbO2 no evidence of decay up to 450 psec was found. Greene et al. (8) conducted a study of HbO2 and HbCO photolysis using 8-psec, 353-nm excitation. They obtained transient absorption spectra in the Soret (400-450 nm) and visible (535-575 nm) regions at 10 psec and 680 psec, finding a persistent, broadened deoxy Hb-like absorption. With their spectra, they observed less than 10% recombination in HbCO over this time interval and less than 20% recombination of photolyzed HbO2. Greene et al. (8) found that HbCO responded similarly to 530-and 358-nm pulses. The present study of the 530-nm photolysis of HbCO and HbO2 is aimed at identifying the species initially generated by light and how these species evolve.Photodissociation of liganded hemoglobins is a well-known phenomenon (1) used to study dynamics of ligand binding to deoxyhemoglobin (Hb) (2)(3)(4)(5)(6)(7)(8). The usefulness of the photoprocess for subsequent dynamical studies is restricted at present by a lack of knowledge of the mechanism of the photodissociation. Of particular interest is the quantum yield for dissociation under steady illumination, which is fairly high (k 0.5) (9, 10) for carboxyhemoglobin (HbCO) but much lower (0 0.05) for the oxy form (HbO2) (9). Two limiting mechanisms can account for the relatively low yield of HbO2 photolysis. First, energy relaxation to nonreactive states may be much faster than the dissociation. Second, although the initial dissociation step in HbO2 may be just as efficient as that in HbCO, some released 02 trapped nearby to the heme may be able to rapidly recombine. These mechanisms are evaluated here by examining the dynamics and identifying the products of the photolysis by picosecond spectroscopy. We have attempted to describe our spectroscopic and dynamical results in terms of recent experimental and theoretical assessments of the excited states of hemoglobins (11-15).The time scale for studying photolysis and subsequent events in heme proteins has in recent years been pressed back to the picosecond regime, starting with the work of Shank et al. (5). Using subpicosecond pulses at 615 nm to excite and probe HbO2 and HbCO, they found induced absorption build-up in less than 0.5 psec for both species, a decay of the HbO2 signal in 2.5 psec, and no decay of the HbCO signal up to 20 psec. Rentzepis and coworkers (6, 7) studied HbCO and the myoglobin compounds MbCO and MbO2 by exciting with 6-psec 530-nm pulses and using 440-nm interrogation over the period 0-00 psec. That group reported a significantly longer (11 psec) predissociation lifetime...

Atomic force microscopy (AFM) was used to image fibrillar and monomeric type I collagen. Protocols were developed to produce samples of air dried fibrillar and monomeric collagen suitable for AFM analysis. Pepsin-extracted, type I bovine skin collagen was polymerized on mica substrates in pH 7.4 phosphate-buffered saline at 37 °C. Monomeric collagen was adsorbed onto mica substrates in 0.012 N HCl or pH 3 buffer at 21 °C. Control samples of buffer-treated mica were also prepared. AFM images of the fibrillar collagen showed well-defined D banding of the fibrils, with a period of 70 nm. Beneath the fibrils finer fibrillar material was seen, probably oligomeric collagen. Samples prepared at acidic pH showed a meshwork of monomeric collagen with an occasional structure of oligomeric size. Control samples of mica treated with buffer only had occasional tree structures formed of crystallized buffer salts. Collagen is an important component of animal tissues and forms extracellular matrix material in combination with other proteins and glycoproteins. The studies reported here form the basis for examination of more complex collagen-containing structures of biological importance.

Picosecond transient absorption spectra of Mb, MbCO, and MbO2 have been studied at time delays.of up to 10 ns after excitation at 353 nm. Particular attention has been paid to the rapid spectral changes that occur in the Soret region during the first 50 ps in MbCO and MbO2. In MbCO both the bleaching of the Soret peak (feature I) and the appearance of new deoxy-like absorption (feature II) occur instantaneously, whereas in MbO2 feature II is delayed with respect to feature I. A short-lived ('=12 ps) feature near 455 nm (feature III) was much more intense in MbO2 than in MbCO and was also identified in the transient spectrum of Mb. No evidence of subnanosecond geminate recombination was found in either MbCO or MbO2. These observations are consistent with a scheme in which MbO2 photodissociates through an excited state of Mb, whereas MbCO under the same conditions produces ground state Mb directly. The results and conclusions are compared with those of previous picosecond studies on these molecules and related hemoglobin derivatives.Picosecond transient absorption spectroscopy is a valuable method for the study ofheme proteins and their reactions with 02 and CO. The unique feature of this technique is its ability to probe changes occurring on a time scale inwhich any largescale movements of the protein are unlikely. In the case of hemoglobin, this permits the observation of the primary processes that initiate the protein conformational changes associated with cooperativity. Understanding these primary processes requires a detailed knowledge of the electronic states of this system and the couplings and interactions between them. To this end, a great deal of experimental and theoretical work has been devoted to establishing an accurate mapping of the states ofvarious hemoglobin derivatives (1). We recently reported the results of a series of picosecond transient measurements on HbCO and HbO2, in which we showed that relaxation pathways derived from spin-orbit coupling arguments could be used to explain the observed spectral changes (2). That work showed how such considerations could account for the difference in the behavior ofthese two molecules: HbO2 exhibited a fast transient (<90 ps) and subnanosecond geminate recombination, whereas in HbCO both of these effects were absent.In order to examine further the scheme proposed in these hemoglobin studies, we have used our picosecond transient absorption spectrometer to study a closely related set ofmyoglobin derivatives: MbCO, MbO2, and unliganded Mb. Myoglobin has a similar electronic structure to hemoglobin but small spectral shifts result from the differences in protein environment. Because of its resemblance to a single hemoglobin subunit, myoglobin provides a natural model for the tetrameric protein, and it has been the object of previous kinetic studies in both picosecond (3,4) and longer time regimes (5, 6). The spectral and temporal information provided by our picosecond spectrometer makes possible a more detailed investigation of the primary photolyt...

We measured picosecond transient resonance Raman spectra of t-stilbene in hexane using a high repetition rate, amplified, synchronously pumped dye laser. We assign four vibrational frequencies to the olefinic moiety. Two of these frequencies, 1238 and 1565 cm−1, have broad, asymmetric bands. We attribute this band shape to a distribution of excited state molecular conformers having different single and double bond strengths.

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