Studies of human hemoglobin intermediates. The double mixing method for studying the reactions of the species Hb4(CO) and Hb4(CO)2.

Using the double mixing method we have studied the reactions of the partially liganded species (Hb4, Hb4L1, Hb4L2, Hb4L3) of normal human hemoglobin with carbon monoxide. In the first mixing, oxygen is removed from the species Hb4(O2) chi (CO) gamma and at the second mixing the species Hb4(CO) gamma reacts with CO. At 90% saturation of oxyHb with CO the main intermediate species are Hb4(CO)3 and Hb4(CO)2, and at 10% saturation Hb4 and Hb4(CO). The four CO-combination rate constants determined are: l'1 = 1 X 10(5) M-1 S-1, l'2 = 7 X 10(5) M-1 S-1, l'3 = 2 X 10(5) M-1 S-1 and l'4 = 4.8 X 10(6) M-1 S-1. The results indicate that there is no monotonic increase in the successive CO-combination rate constants. It is difficult to explain these results on the basis of the two-state model (Monod et al., 1965) or the stereochemical model of Perutz (1970).

Geminate recombination in carboxy hemoglobin A and its relation to overall carbon monoxide reactivity.

The geminate recombination of CO with carboxy hemoglobin (Hb4(CO)3) following a ten nanosecond laser pulse and the overall combination of the fourth CO with Hb4(CO)3 has been studied as a function of pH in the presence and absence of inositol hexaphosphate. The results indicate that the kinetics of both reactions are independent of pH and phosphate concentration. The results are discussed in terms of a two-step mechanism: a pre-equilibrium step followed by heme--ligand bond formation. The latter is also known as the geminate recombination reaction (Hb + CO in equilibrium Hb X CO in equilibrium HbCO).

A general computer approach for calculating rate constants from near-equilibrium kinetic studies.

A general computer approach for estimating rate constants from relaxation times is described. The programme CORNEK is essentially a least-squares refinement programme applied to non-linear systems. It uses directly the differential forms of the first derivatives of mass-balance and rate equations, thus avoiding the time-consuming derivations of near-equilibrium rate equations. The programme has been tested for binary systems such as Cu-histamine, Cu-serine, and the ternary system Cu-histamine-serine.

Comparing hand hygiene measures in a neonatal ICU: a randomized crossover trial.

OBJECTIVE: To compare plain soap, alcohol hand rub and iodophors as hand hygiene measures in a neonatal intensive care unit (NICU). DESIGN: Randomized, crossover, three-armed, controlled trial with blinded outcome measurement. SETTING: Level III NICU. PARTICIPANTS: 35 NICU nurses. INTERVENTION: Participants were assigned to plain soap hand washing, alcohol hand rub and povidone-iodine hand scrub by a random cross-over design. Interventions were preceded by 14-day neutral periods. Cultures from hands were taken before and after each hand-hygiene use, prior to 5 patient-care activities. MAIN OUTCOME MEASURE: The primary outcome was mean post-hygiene colony forming unit count (CFU-C). RESULTS: There were differences between soap, alcohol and povidone groups vis a vis posthygiene CFUC [median: 60, 8 and 10.5, respectively (P<0.001)], absolute reduction in CFU-C [median: 15, 100 and 40, respectively (P<0.001)], percent reduction in CFUC [median: 33.3, 92 and 87, respectively (P<0.001)] and proportion with low CFU-C [47%, 71% and 72%, respectively (P<0.001)]. Alcohol [Adjusted OR 3.2 (95% CI 1.9, 5.3)], povidone-iodine [AOR 3.1 (95% CI 1.8, 5.3)] and high prehygiene CFU-C (>300) [AOR 0.18 (95% CI 0.1, 0.3)] were independently associated with low CFU-C. CONCLUSION: After a 2 minute hand wash at entry into NICU, alcohol hand rub and povidone-iodine scrub are superior to plain soap hand wash for subsequent decontamination of hands of nurses working in NICU.

Kinetic studies on partially liganded species of carboxyhemoglobin: (alpha 1CO-beta 1CO)alpha 2 beta 2 or (alpha 2CO beta 2CO)alpha 1 beta 1.

Kinetics of CO combination with and dissociation from isomer III, (alpha 1CO beta 1CO)alpha 2 beta 2 or alpha 1 beta 1 (alpha 2CO beta 2CO), and Hb Rothschild have been studied using the double mixing and microperoxidase methods. Isomer III was prepared in a manner so that it was the only reactive species in the reaction mixture. The biphasic reaction time course in both the "on" and "off" reactions of isomer III and the CO combination reaction of Hb Rothschild are attributed to slow relaxation between the fast and slow CO-reacting species in the two proteins: isomer III: l'f = 6 x 10(6) M-1 s-1, l'dimer = 1.7 x 10(6) M-1 s-1, l's = 2.2 x 10(5) M-1 s-1, lf = 0.15 s-1, ls = 0.01 s-1; Hb Rothschild: l'f = 2.8 x 10(6) M-1 s-1; l's = 2.7 x 10(5) M-1 s-1.

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO.

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.