N-Cα Bond Cleavage of Zinc-Polyhistidine Complexes in Electron Transfer Dissociation Mediated by Zwitterion Formation: Experimental Evidence and Theoretical Analysis of the Utah-Washington Model.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) of gas-phase ions are widely used for peptide/protein sequencing by mass spectrometry. To understand the general mechanism of ECD/ETD of peptides, we focused on the ETD fragmentation of metal-peptide complexes in the absence of remote protons. Since Zn(2+) strongly binds to neutral histidine residues in peptides, Zn(2+)-polyhistidine complexation does not generate any remote protons. However, in the absence of remote protons, electron transfer to the Zn(2+)-polyhistidine complex induced the N-Cα bond cleavage. The formation pathway for the ETD products was investigated by density functional theory calculations. The calculations showed that the charge-reduced zinc-peptide radical, [M + Zn](•+), can exist in the low-energy zwitterionic amide π* states, which underwent homolytic N-Cα bond dissociation. The homolytic cleavage resulted in the donation of an electron from the N-Cα bond to the nitrogen atom, producing an iminoenol c' anion. The counterpart z(•) radical contained a radical site on the α-carbon atom. The iminoenol c' anion then abstracted a proton to presumably form the more stable amide c' fragment. The current experimental and computational joint study strongly suggested that the N-Cα bond cleavage occurred through the aminoketyl radical-anion formation for Zn(2+)-polyhistidine complexes in ETD.

Influence of metal-peptide complexation on fragmentation and inter-fragment hydrogen migration in electron transfer dissociation.

The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissociation (ETD) in tandem mass spectrometry. In the present study, K(+) and Ca(2+) were used as charge carriers to form multiply-charged metal-peptide complexes. ETD of the potassium- or calcium-peptide complex was initiated by transfer of an electron to a proton remote from the metal cation, and a c'-z• fragment complex, in which the c' and z• fragments were linked together via a metal cation coordinating with several amino acid residues, was formed. The presence of a metal cation in the precursor for ETD increased the lifetime of the c'-z• fragment complex, eventually generating c• and z' fragments through inter-fragment hydrogen migration. The degree of hydrogen migration was dependent on the location of the metal cation in the metal-peptide complex, but was not reconciled with conformation of the precursor ion obtained by molecular mechanics simulation. In contrast, the location of the metal cation in the intermediate suggested by the ETD spectrum was in agreement with the conformation of "proton-removed" precursors, indicating that the charge reduction of precursor ions by ETD induces conformational rearrangement during the fragmentation process.

Physiological responses of simulated karate sparring matches in young men and boys.

The purpose of this study was to investigate the duration of each series of offensive and defensive techniques and the cardiovascular, metabolic, and perceptual responses during 2- and 3-minute bouts of simulated karate sparring. Six young men (age, 18-20 years) and 6 boys (age, 16-17 years) participated in this study. We formed 3 pairs of men and 3 pairs of boys to create a demanding competitive environment. After a rest period, each pair performed a 2-minute bout of sparring, sat quietly for 60 minutes, and then performed 3-minute bout of sparring. We measured oxygen uptake (Vo2), heart rate (HR), and blood lactate responses and ascertained the rate of perceived exertion (RPE) and energy expenditure (EE) during these sparring bouts. The ventilatory threshold was estimated from ventilatory equivalent and Vo2 obtained during the treadmill test. The duration of each series of offensive and defensive techniques was videotaped. During the 2- and 3-minute bouts of sparring, the duration of longest series of offensive and/or defensive combination techniques performed were 2.1 +/- 1.0 and 1.8 +/- 0.4 seconds, respectively; the mean total times of performing offensive and defensive techniques were 13.3 +/- 3.3 and 19.4 +/- 5.5 seconds, respectively. The mean oxygen uptake (Vo2), the percentage of maximum oxygen uptake (%Vo2max), HR, percentage of maximum HR, RPE, and EE for a 3-minute bout of sparring were significantly higher than for a 2-minute bout of sparring. The mean %Vo2max values for these bouts of sparring were below the ventilatory threshold. It is recommended that karate practitioners perform more specific weight training, plyometric exercises, and interval training to increase the ability to buffer acid muscle and blood concentrations and to build lean body mass, strength, and power to develop the specific motor skills required in sparring.