Time-resolved absorption and UV resonance Raman spectra reveal stepwise formation of T quaternary contacts in the allosteric pathway of hemoglobin.

Hemoglobin undergoes a series of molecular changes on the nanosecond and microsecond time-scale following photodissociation of CO ligands. We have monitored these processes with a combination of transient absorption and resonance Raman (RR) spectroscopy. The latter have been acquired at higher data rates than previously available, thanks to kilohertz Ti:sapphire laser technology, with frequency-quadrupling into the ultraviolet. As a result of improved resolution of the UVRR time-course, a new intermediate has been identified in the pathway from the R (HbCO) to the T (deoxyHb) state. This intermediate is not detected via absorption transients, since the change in heme absorption is insignificant, but its lifetime agrees with a reported magnetic circular dichroism transient, which has been attributed to a quaternary tryptophan interaction. The new UVRR data allow elaboration of the allosteric pathway by establishing that the T-state quaternary contacts are formed in two well-separated steps, with time constants of 2.9 micros and 21 micros, instead of a single 20 micros process. The first step involves the "hinge" region contacts, as monitored by the Trp beta 37...Asp alpha 94 H-bond, while the second involves the "switch" region, as monitored by the Tyr alpha 42...Asp beta 99 H-bond. A working model for the allosteric pathway is presented.

Shear effects on aluminum phosphate adjuvant particle properties in vaccine drug products.

Adjuvant-containing drug products can be exposed to high levels of interfacial shear during manufacture. This may affect the integrity of the adjuvant, alter its interaction with the drug substance or change the physical characteristics of the drug product. In this study, a solid-liquid interfacial shear device was used to investigate the shear response of aluminum phosphate adjuvant alone and two adjuvant containing vaccine drug products (DP1 and DP2). The relationship between the shear sensitivity of each and its resuspension properties was determined. Changes in the particle dimensions of the bulk adjuvant were minimal at shear strain rates of 10,900 s(-1) . However, at 25,500 s(-1) , the median particle diameter was reduced from 6.2 to 3.5 µm and was marked by the presence of sub-micron fines. A formulation without drug substance and DP2 produced similar shear responses but with less impact on particle diameter. The behavior of DP1 was less predictable. Sheared DP1 was characterized by prolonged sedimentation because of the presence of fine particulates and required in excess of 300 rotations to resuspend after extended storage. The study confirms that the solid-liquid interfacial shear device may be applied to understand product shear sensitivity associated with vaccine manufacturing.

Cross-linking of gelatin capsules with formaldehyde and other aldehydes: an FTIR spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy (FTIR) has been used to study cross-linking in hard gelatin capsules induced by exposure to formaldehyde, acetaldehyde, and propionaldehyde. These aldehydes are known to cause cross-linking between the amino acid chains of gelatin. Using FTIR spectroscopy, it is possible to analyze the cross-linking mechanisms by studying changes in the vibrational bands of the gelatin spectrum. The FTIR spectrum changes over time when the capsules are left in an aldehyde-rich environment. Analysis of the spectra shows that the early observed spectral changes conform to reaction intermediates proposed in previous work based on nuclear magnetic resonance experiments, specifically, the formation of amine methyl alcohol of arginine and lysine residues. Further spectral changes appear to be mostly from unreacted aldehydes absorbed to the gelatin, although a minor shift of the amide II peak is attributed to cross-link formation.

Dynamics of carbon monoxide binding to CooA.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.