Role of early life nutrition on regulating the hypothalamic­anterior pituitary­testicular axis of the bull

The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein­Friesian bull calves. Holstein­Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a HIGH (n = 10) or LOW (n = 10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 ± 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein­Friesian bulls (n = 10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P< 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P < 0.001), had larger testes (P < 0.001), larger seminiferous tubule diameter (P < 0.001), more mature spermatogenic cells (P < 0.001) and more Sertoli cells (P < 0.05) in accordance with both morphological and transcriptional data. Overall, testicular gene expression profiles suggested a more mature stage of development in HIGH compared with LOW and were more closely aligned to that of mature bulls. Ghrelin receptor was the only differentially expressed gene between LOW and HIGH calves in either the anterior pituitary (P < 0.05) or arcuate nucleus region of the hypothalamus (P < 0.10) and was upregulated in LOW for both tissues. This study indicates that an enhanced plane of nutrition during early calfhood favourably alters the biochemical regulation of the hypothalamus­anterior pituitary­testicular axis, advancing testicular development and hastening spermatogenesis.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: I. Effects on performance, body composition, age at puberty, and postpubertal semen production.

The aim of this study was to examine the effect of plane of nutrition (1) during the first 6 mo of life and (2) from 6 mo of age to puberty on early growth characteristics, age at puberty, and postpubertal semen production in Holstein-Friesian bulls. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high (Hi) or low (Lo) plane of nutrition for the first 6 mo of life. The Hi and Lo calves received 1,200 and 450 g of milk replacer, respectively; Hi calves were fed concentrate ad libitum and Lo were fed a maximum of 1 kg concentrate daily, and concentrate allowances remained the same after weaning. At 24 wk of age, bulls were reassigned within treatment to either remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: Hi-Hi, Hi-Lo, Lo-Lo, and Lo-Hi. After puberty, all bulls were fed a moderate plane of nutrition until 60 wk of age; thereafter, the diet was ad libitum concentrates until slaughter at 72 wk of age. Bulls were weighed weekly before weaning and every 2 wk after weaning. Scrotal circumference (SC) was measured every 2 wk, beginning at 15 wk of age. Beginning at a SC of 24 cm, electro-ejaculation was carried out every 2 wk to establish the onset of puberty. Semen collection continued monthly after puberty. Thermal images of the scrotum were taken monthly from 28 to 36 wk of age. Scrotal skin thickness (SST) was measured monthly (from 16 wk of age to puberty) using a digital calipers. Bulls on the Hi diet had a higher scrotal temperature and SST at each time point than those on the Lo diet. Average daily gain (ADG) was greatest in Hi-Hi bulls, with Hi-Lo and Lo-Hi having similar ADG but both being greater than Lo-Lo. Bulls on the Hi diet pre-6 mo of age were younger at puberty, regardless of diet offered post-6 mo of age. Bulls offered a Hi diet post-6 mo were heavier at puberty. Neither scrotal temperature nor dietary treatment affected postpubertal semen production variables. In conclusion, a high plane of nutrition during the first 6 mo of age hastened the onset of puberty and the availability of saleable semen, regardless of plane of nutrition post-6 mo of age.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: II. Effects on metabolic and reproductive endocrinology and identification of physiological markers of puberty and sexual maturation.

The aim of this study was (1) to examine the effect of plane of nutrition during the first and second 6 mo of life on systemic concentrations of reproductive hormones and metabolites in Holstein-Friesian dairy bulls, and (2) to establish relationships with age at puberty and postpubertal semen production potential. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high or low plane of nutrition for the first 6 mo of life. At 24 wk of age, bulls were reassigned, within treatment, either to remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: high-high, high-low, low-low, and low-high. Monthly blood samples were analyzed for metabolites (albumin, urea, total protein, ß-hydroxybutyrate, glucose, nonesterified fatty acid, triglycerides and creatinine), insulin, insulin-like growth factor-1, leptin, adiponectin, FSH, and testosterone. A GnRH challenge was carried out at 16 and 32 wk of age (n = 9 bulls per treatment). Blood was collected at 15-min intervals for 165 min, with GnRH administered (0.05 mg/kg of body weight, i.v.) immediately after the third blood sample. Blood samples were subsequently analyzed for LH, FSH, and testosterone. Stepwise regression was used to detect growth and blood measurements to identify putative predictors of age at puberty and subsequent semen quality traits. Metabolic hormones and metabolites, in general, reflected metabolic status of bulls. Although FSH was unaffected by diet, it decreased with age both in monthly samples and following GnRH administration. Testosterone was greater in bulls on the high diet before and after 6 mo of age. Testosterone concentrations increased dramatically after 6 mo of age. Luteinizing hormone was unaffected by diet following GnRH administration but basal serum LH was greater in bulls on a high diet before 6 mo of age. In conclusion, the plane of nutrition offered before 6 mo of age influenced metabolic profiles, which are important for promoting GnRH pulsatility, in young bulls.

Effect of Early Calf-Hood Nutrition on the Transcriptional Regulation of the Hypothalamic-Pituitary-Testicular axis in Holstein-Friesian Bull Calves.

The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of the arcuate nucleus of the hypothalamus, anterior pituitary and testes in Holstein-Friesian bulls. Holstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were offered a high (n = 10) or low (n = 10) plane of nutrition in order to achieve an overall growth rate of 1.2 and 0.5 kg/day. At 126 (±3) days of age, calves were euthanized, hypothalamus (arcuate region), anterior pituitary and testicular parenchyma samples were harvested and RNAseq analysis was performed. There were 0, 49 and 1,346 genes differentially expressed in the arcuate nucleus, anterior pituitary and testicular tissue of bull calves on the low relative to the high plane of nutrition, respectively (P < 0.05; False Discovery Rate <0.05). Cell cycle processes in the anterior pituitary were down regulated in the low relative to the high plane of nutrition; there was no differential expression of genes related to reproductive processes. Gene expression involved in cholesterol and androgen biosynthesis in the testes were down regulated in animals on the low plane of nutrition. This study provides insight into the effect of early life plane of nutrition on the regulation of the HPT axis.

Effect of milk replacer and concentrate intake on growth rate, feeding behaviour and systemic metabolite concentrations of pre-weaned bull calves of two dairy breeds.

Early-life nutrition affects calf development and thus subsequent performance. The aim of this study was to examine the effect plane of nutrition on growth, feeding behaviour and systemic metabolite concentrations of artificially reared dairy bull calves. Holstein-Friesian (F; n=42) and Jersey (J; n=25) bull calves with a mean±SD age (14±4.7 v. 27±7.2 days) and BW (47±5.5 v. 33±4.7 kg) were offered a high, medium or low plane of nutrition for 8 weeks using an electronic feeding system which recorded a range of feed-related events. Calves were weighed weekly and plasma samples were collected via jugular venipuncture on weeks 1, 4 and 7 relative to the start of the trial period. The calves offered a high plane of nutrition had the greatest growth rate. However, the increased consumption of milk replacer led to a reduction in feed efficiency. Holstein-Friesian calves offered a low plane of nutrition had the greatest number of daily unrewarded visits to the feeder (P<0.001). ß-hydroxybutyrate (BHB) concentrations were greater in F calves on a low plane of nutrition (P<0.001). Although there was no effect of plane of nutrition, BHB concentrations in F calves increased before weaning, concomitant with an increase in concentrate consumption. Urea concentrations were unaffected by plane of nutrition within either breed. Jersey calves on a low plane of nutrition tended to have lower triglycerides than those on a high plane (P=0.08), but greater than those on a medium plane (P=0.08). Holstein-Friesian calves offered a high plane of nutrition tended to have greater triglyceride concentrations than those on a medium plane (P=0.08). Triglycerides increased from the start to the end of the feeding period (P<0.05), across both breeds. A medium plane of nutrition resulted in a growth, feeding behaviour and metabolic response comparable with a high plane of nutrition in pre-weaned bull calves of both F and J breeds.

Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality.

The aim of this study was to examine the effects of dietary supplementation with rumen protected n-6 or n-3 polyunsaturated fatty acids (PUFA) on the quantity and quality of semen from young post-pubertal dairy bulls. Pubertal Holstein-Friesian (n = 43) and Jersey (n = 7) bulls with a mean ± s.e.m. age and bodyweight of 420.1 ± 5.86 days and 382 ± 8.94 kg, respectively, were blocked on breed, weight, age and semen quality (based on the outcomes of two pre-trial ejaculates) and randomly assigned to one of three treatments: (i) a non-supplemented control (CTL, n = 15), (ii) rumen-protected safflower (SO, n = 15), (iii) rumen-protected n-3 PUFA-enriched fish oil (FO, n = 20). Bulls were fed their respective diets, ad libitum for 12 weeks; individual intakes were recorded using an electronic feeding system for the initial 6 weeks of the feeding period. Semen was collected via electro-ejaculation at weeks -2, -1, 0, 7, 10, 11 and 12 relative to the beginning of the trial period (week 0). On collection, semen volume, sperm concentration and progressive linear motility (PLM) were assessed. On weeks -2, -1, 0, 10, 11, 12, semen was packaged into 0.25 mL straws and frozen using a programmable freezer. On weeks -1, 7 and 11; a sub-sample of semen was separated into sperm and seminal plasma, by centrifugation and stored at - 20 °C until analysis of lipid composition. Semen from 10 bulls per treatment were used for post-thaw analysis at weeks 10, 11 and 12 (3 straws per ejaculate). Sperm motility was analysed by computer assisted semen analysis (CASA). In addition, membrane fluidity, acrosome reaction and oxidative stress were assessed using flow cytometry. Sperm from bulls fed SO had a 1.2 fold higher total n-6 PUFA content at week 11 compared to week -1 (P < 0.01) while bulls fed FO had a 1.3 fold higher total n-3 PUFA content, in sperm by week 11 (P < 0.01). There was no effect of diet on semen volume, concentration or PLM of sperm when assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls.

Effect of breed, plane of nutrition and age on growth, scrotal development, metabolite concentrations and on systemic gonadotropin and testosterone concentrations following a GnRH challenge in young dairy bulls.

The onset of puberty in the bull is regulated by the timing of early GnRH pulsatility release from the hypothalamus, which has been demonstrated to be affected by plane of nutrition during calf-hood. The aim of this study was to determine the effect of plane of nutrition on growth rate, scrotal development, metabolite concentrations and exogenous gonadotrophin (GnRH) induced release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (TT) in pre-pubertal bulls of two contrasting dairy breeds. Holstein-Friesian and Jersey bull calves were assigned to either a high or low plane of nutrition from 3 to 49 weeks of age. Intensive blood sampling was conducted at 16, 24 and 32 weeks of age, every 15 min from 30 min prior to intravenous administration of exogenous GnRH to 135 min after. Monthly blood samples were also collected and analyzed for insulin like growth factor 1 (IGF-1), insulin, leptin, adiponectin and metabolite concentration. Insulin and IGF-1 were higher in bulls on a high plane of nutrition (P < 0.001) but were not affected by breed (P > 0.05). Leptin was not affected by plane of nutrition or breed (P > 0.05). Adiponectin tended to be higher in bulls on a high plane of nutrition (P = 0.05), but was not affected by breed (P > 0.05). Bulls on a high plane of nutrition had a greater concentration of LH in response to GnRH (P < 0.05) but there was no effect of breed (P > 0.05). FSH concentration was not influenced by breed or plane of nutrition but FSH concentrations did decrease with age (P < 0.01), while, LH was not affected by age (P > 0.05). Jersey bulls, particularly those on a high plane of nutrition, had higher TT production in the pre-pubertal period (P < 0.001). Using 28 cm as a proxy for age at puberty, bulls on a high plane of nutrition were predicted to reach puberty earlier than bulls on a low plane. In conclusion, the data clearly demonstrate that a high plane of nutrition positively affects several key nutritional and reproductive hormones which are critical to the endocrinological functionality of the hypothalamic-pituitary-testicular axis in dairy-bred bull calves.

Targeted proteomics identify metabolism-dependent interactors of yeast cytochrome c peroxidase: implications in stress response and heme trafficking.

Recently we discovered that cytochrome c peroxidase (Ccp1) functions primarily as a mitochondrial H2O2 sensor and heme donor in yeast cells. When cells switch their metabolism from fermentation to respiration mitochondrial H2O2 levels spike, and overoxidation of its polypeptide labilizes Ccp1's heme. A large pool of heme-free Ccp1 exits the mitochondria and enters the nucleus and vacuole. To gain greater insight into the mechanisms of Ccp1's H2O2-sensing and heme-donor functions during the cell's different metabolic states, here we use glutathione-S-transferase (GST) pulldown assays, combined with 1D gel electrophoresis and mass spectrometry to probe for interactors of apo- and holoCcp1 in extracts from 1 d fermenting and 7 d stationary-phase respiring yeast. We identified Ccp1's peroxidase cosubstrate Cyc1 and 28 novel interactors of GST-apoCcp1 and GST-holoCcp1 including mitochondrial superoxide dismutase 2 (Sod2) and cytosolic Sod1, the mitochondrial transporter Pet9, the three yeast isoforms of glyceraldehyde-3-phosphate dehydrogenase (Tdh3/2/1), heat shock proteins including Hsp90 and Hsp70, and the main peroxiredoxin in yeast (Tsa1) as well as its cosubstrate, thioreoxin (Trx1). These new interactors expand the scope of Ccp1's possible roles in stress response and in heme trafficking and suggest several new lines of investigation. Furthermore, our targeted proteomics analysis underscores the limitations of large-scale interactome studies that found only 4 of the 30 Ccp1 interactors isolated here.

NG-monomethyl-L-arginine co-injection attenuates the thermogenic and hyperthermic effects of E2 prostaglandin microinjection into the anterior hypothalamic preoptic area in rats.

Prostaglandin E2 (PGE2) microinjection (25 ng, 250 nl) into the preoptic area of the anterior hypothalamus (POAH) stimulated heat production in brown adipose tissue (BAT) and increased core temperature in urethane-anesthetized rats. These thermogenic and hyperthermic effects were attenuated by co-injection of NG-monomethyl-L-arginine (NMMA, 25 micrograms), a competitive inhibitor of nitric oxide (NO) production from L-arginine. Inclusion of L-arginine (50 micrograms), though not D-arginine (50 micrograms) reversed the inhibitory effect of NMMA (25 micrograms) on intra-POAH PGE2-induced increases in interscapular BAT (IBAT) and core temperatures. Intra-POAH injection of NMMA (25 micrograms) or L-arginine (50 micrograms) alone had no effect on IBAT and core temperatures. The results suggest that the effect on thermoregulation induced by action of PGE2 in the POAH is modulated by a local L-arginine-dependent and NMMA-sensitive NO-generating system.

An inhibitor of nitric oxide production, NG-nitro-L-arginine-methyl ester, improves survival in anaphylactic shock.

Induction of anaphylactic shock in mice by i.v. antigen challenge (bovine serum albumin, 100 micrograms) or i.v. treatment with the mast cell degranulator compound 48/80 resulted in 80 and 90% mortality rate, respectively. Inhibition of nitric oxide (NO) synthesis from L-arginine by co-injection of the L-arginine analog NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) reduced the mortality rate by 40 and 20% in the antigen- and compound 48/80-induced shock models. Treatment with 60 mg/kg L-NAME reduced the mortality rate by 60% in these shock models. This beneficial effect was reversed by addition of L-arginine (120 mg/kg) but not D-arginine (120 mg/kg). These results suggest NO production as a possible mechanism involved in the pathophysiology of anaphylactic shock.

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH.

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP (MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP (MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP (MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda CT = 637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm.

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-ß-cyclodextrin-cholesterol (MßCD)/mL (MßCD0.75); TALP+1.5mg MßCD-cholesterol/mL (MßCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MßCD1.5 and MßCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MßCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MßCD1.5 and MßCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation.

Functional anatomy of the hands of fur seals and sea lions.

Dissections and manipulations of the hands of 14 specimens of four genera of fur seals and sea lions and of generalized fissiped carnivores were used to identify the structural modifications involved in formation of the expanded forelimb paddles characteristic of these pinnipeds. The morphological peculiarities were then correlated with differences in locomotor function, both on land and in the water, on the basis of previously published data, for the purpose of identifying modifications of structure and function which have adaptive vale. Structural differences found in the manus of fur seals and sea lions include: (1) reduction in size of the ulnar side of the carpus and a radial shift in the length-order of the digits, (2) development of musculature in the antebrachial fascia which attaches to the caudal margin of the flipper, (3) orientation of the radial side of the manus dorsal and radial to the rest of the hand, (4) increased range of possible midcarpal movement and in deviational mobility at the first and fifth digits, (5) attachment of forearm musculature onto radial digits and (6) well-developed hypothenar muscles and absence of thenar muscles. Modifications in hand structure are viewed as providing a morphological basis for employment of the hand to advantage during aquatic locomotion while maintaining thrust-producing and potentially energy-conserving capacities during movement on land. As such these differences in structure and function are viewed as adaptations to locomotion in the water and on land which are extensions of locomotor adaptations attributed to modifications of forelimb structure and function associated with the generation of massive aquatic locomotor thrust. The adaptive value of such modifications is interpreted as allowing fur seals and sea lions to swim with speed and thereby capture elusive fast-swimming prey while maintaining a level of terrestrial locomotor ability compatible with their amphibious mode of life.

Probing local thermal stabilities of bovine, horse, and tuna ferricytochromes c at pH 7.

Correlation between the flexibility of the Met80 loop (residues 75-86) and the local stabilities of native ferricytochromes c from horse, bovine, and tuna was examined. By monitoring the heme bands versus temperature, absorption changes associated with altered ligation in the alkaline isomers were observed. In addition, the intensity of the 695-nm absorption band, which is associated with the heme-crevice stability, decreased with increasing temperature and exhibited biphasic temperature dependence, with transition temperatures (Tc) at 35 degrees C in tuna c, 55 degrees C in horse c, and 58 C in bovine c. Since the heme crevice plays a key role in the thermal stabilities of cytochromes c, their susceptibility to proteolytic attack was examined as a function of temperature. Proteolytic digestion, which requires local conformational instability, revealed that the local stabilities of the cytochromes follow the order: bovine > horse >> tuna, and increased digestion occurred at temperatures close to the 695-nm Tc for each protein. This is consistent with the actual substitution of the Met80 ligand above the 695-nm Tc, which is reflected in the thermodynamic parameters for the two phases. Also, tuna c, unlike horse and bovine c, exhibits different 695-nm (35 degrees C) and Soret (approximately 46 degrees C) Tc values, but its local stability is controlled by the transition detected at 695 nm. The combined spectroscopic and proteolysis results clearly indicate that the flexibility of the Met80 loop determines the local stability of cytochromes c.

The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt.

Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.

Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping.

Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H(2)O(2) were investigated by electrospray mass spectrometry (ESI-MS) using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS). Up to four DBNBS-cyt c adducts were observed in the mass spectra. However, by varying the reaction conditions (0-5 molar equivalents of H(2)O(2) and substituting cyt c with its cyanide adduct which is resistant to peroxidation), noncovalent DBNBS adduct formation was inferred. Nonetheless, optical difference spectra revealed the presence of a small fraction of covalently trapped DBNBS. To probe the nature of the noncovalent DBNBS adducts, the less basic proteins, metmyoglobin (Mb) and alpha-lactalbumin, were substituted for cyt c in the cyt c/H(2)O(2)/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H(2)O(2)/DBNBS reactions, whereas no adducts were detected following alpha-lactalbumin/H(2)O(2)/DBNBS incubation, which is consistent with adduct formation via spin trapping only. Titration with DBNBS at pH 2.0 yielded noncovalent DBNBS-cyt c adducts and induced folding of acid-denatured cyt c, as monitored by ESI-MS and optical spectroscopy, respectively. Thus, the noncovalent DBNBS-cyt c mass adducts observed are assigned to ion pair formation occurring between the negatively charged sulfonate group on DBNBS and positively charged surface residues on cyt c. The results reveal the pitfalls inherent in using mass spectral data with negatively charged spin traps such as DBNBS to identify sites of radical formation on basic proteins such as cyt c.

Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH.

Despite highly conserved active-site structures, members of the plant peroxidase superfamily exhibit a wide range of pH optima. Horseradish peroxidase isozyme C (HRPC) is an ideal peroxidase to investigate the structural determinants of pH stability and activity in superfamily members. Conflicting reports exist on the low-pH stability of HRPC and consequently the pKa of the catalytic distal histidine, which is neutral in active peroxidases. Towards resolving such discrepancies, acid-induced changes in HRPC from two popular commercial suppliers were systematically analyzed. Specifically, FTIR v(CO) and Soret-CD spectra of HRPC-CO and Soret absorption of ferric HRPC were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers, while the FTIR amide I' and far-UV CD spectra were examined to probe changes in secondary structure. Both HRPC-CO samples exhibited identical pH 7.0 v(CO) bands at 1934 and 1905 cm-1. In the pH 3.0 spectrum of sample A, the 1934 cm-1 band was dominant while a broad 1969 cm-1 band appeared in sample B. The intensity of this band, which is assigned to solvent-exposed heme, was greater in citrate than phosphate buffer, but in formate the 1934 cm-1 band remained dominant. Other spectral changes mirrored the v(CO) trends. No time- or buffer-anion-dependent conformation changes were detected in 1 mM CaCl2, revealing that buffer-anion-dependent leaching of stabilizing Ca2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation, which was detected by SDS-PAGE. It is further proposed that glycosylation patterns may affect the low-pH stability of class II and III plant peroxidases.

Fluorescence investigation of yeast cytochrome c peroxidase oxidation by H2O2 and enzyme activities of the oxidized enzyme.

The role of tryptophan residues as endogenous electron donors in cytochrome c peroxidase (CCP) was examined by protein steady-state fluorescence. Compound I and more highly oxidized forms of CCP were formed by adding 1, 3, and 10 equiv of H2O2 to 5 microM protein at pH 7.0 in the absence of exogenous reducing substrates. Addition of native CCP to 8 M urea at pH 1.5 relieved heme quenching, and compound I exhibited 90 +/- 4% fluorescence relative to unoxidized CCP, consistent with the loss of 0.7 +/- 0.2 tryptophan and the assignment of the primary radical site to Trp191. CCP oxidized with 10-fold excess H2O2 exhibited 65 +/- 1% relative fluorescence, indicating loss of 2.4 +/- 0.1 tryptophans. Compound I and the higher oxidized forms of CCP spontaneously decayed to ferric CCP species over approximately 24 h with the loss of approximately 0.5 additional tryptophan in each case. The 24-h decay product of compound I exhibited 73% activity, 74% H2O2 titer, and titration led to the further oxidation of approximately 0.6 tryptophan. However, no further tryptophan oxidation was observed on titration of the 24-h decay products of samples initially oxidized with 3 and 10 equiv of H2O2. These samples exhibited 58 and 18% H2O2 titer, and 47 and 16% activity, respectively, which shows that radical formation of Trp191 is not required for activity. The fluorescence decrease with time paralleled the decrease in activity of H2O2-oxidized CCP using both ferrocytochrome c and ferrocyanide as substrates, indicating that tryptophan and activity loss occurred on similar time scales.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy.

Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast [cyt(Y)] and horse heart [cyt(H)] was studied by resonance Raman spectroscopy. On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins. At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74%. In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%). In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation. In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains. The biological significance of the conformational changes in CCP are discussed. In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation.