Jack Hegenauer

Staining acidic phosphoproteins (phosvitin) in electrophoretic gels

The principal phosphoglycoproteins of avian and amphibian egg yolk, known as phosvitins, have been examined extensively in developmental studies of estrogenic induction of protein synthesis (1) and, more recently, as substrates for protein kinases which phosphorylate the basic nuclear proteins (2,3). Phosvitin has few aromatic and basic amino acids (4) which can be detected by uv absorbance or by conventional anionic protein stains following electrophoresis. In addition, the high negative-charge density of clustered phosphorylserine residues (2) may prevent staining by charge repulsion of anionic dyes. Dilute solutions of cationic dyes like toluidine blue and acridine orange have been employed (5,6), but, in our experience, they penetrate gels slowly and also stain the residual negative charges of most electrophoretic matrices, including agarose, starch, and even polyacrylamide. The only specific method for localizing phosphoproteins in polyacrylamide gels requires several time-consuming steps to liberate orthophosphate by basic hydrolysis, to form an insoluble phosphomolybdate complex, and, finally, to stain this complex with methyl green dye (7). The exceptional affinity of contiguous phosphorylserines for trivalent metal ions (8) can, however, be exploited to detect phosvitin in gels. This strategem may also provide an alternative method for visualizing similar acidic phosphoproteins which have now been identified in brain, spermatozoa, and adrenal medulla (9,lO).

Synthesis and structure of a trinuclear chromium(III)-nicotinic acid complex

Abstract The reaction of nicotinic acid (nic), chromic perchlorate hexahydrate, and sodium perchlorate in aqueous solution affords a Cr(III)-vitamin complex whose composition has been established to be Na[Cr 3 O(nicH) 6 (H 2 O) 3 ][ClO 4 ] 8 ·nicH·6H 2 O, based on analytical data and a single-crystal X-ray analysis. In the [Cr 3 O(nicH) 6 (H 2 O) 3 ] +7 ion a central oxygen atom is bonded to three chromium atoms. The nicotinic and zwitterions bridge these chromium centers through the carboxylate oxygen atoms. On each chromium atom a water oxygen, trans to the central oxygen atom, complexes the octahedral coordination. The ion has crystallographically imposed symmetry 6 . The binding of nicotinic acid in this Cr(III)-nicotinic acid complex is significantly different from that currently proposed for the glucose tolerance factor.

Effects of exercise on iron metabolism in rats

Abstract The effect of exercise on the uptake, redistribution, and excretion of iron was studied in normal iron-sufficient rats. Rats were run to exhaustion in a “sprint” protocol seven times over a twentyone day period. Exercised rats did not differ significantly from sedentary rats in hemoglobin concentrations or in body or organ weights. Exercised rats absorbed more iron than sedentary rats in radioiron tracer studies. Radioiron accumulated mainly in heart, liver, and spleen. Strikingly, tracer iron accumulated significantly in hearts of exercised rats. Exercised rats had less total iron in liver and spleen than sedentary rats. Myoglobin concentrations in heart and soleus muscles were significantly higher in rats after the exercise regimen and decreased following fourteen days of rest. Brief, strenuous exercise appears to signal increased absorption of iron and to stimulate the redistribution of storage iron. This mobilization of iron during exercise may be directed towards the enhancement of oxygen-accepting ability in muscles at work.

Metal-ion-directed site-specificity of hydroxyl radical detection

A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.

Transitory hematologic effects of moderate exercise are not influenced by iron supplementation

SummaryA young womens exercise/fitness class tested the idea that administration of supplemental iron would prevent “sports anemia” that may develop during exercise and training and improve iron status of exercising females of menstrual age. Fifteen women (aged 18–37) were selected for each of three treatment groups: (1) no supplemental iron; (2) 9 mg·d−1 of Fe; and (3) 18 mg·d−1 of Fe (1 US Recommended Daily Allowance). Women exercised at approximately 85% of maximal heartrate for progressively increasing lengths of time in a jogging program and worked up to 45 min of exercise 4 d·week−1 for 8 weeks. Hematologic analysis was performed in weeks 1, 5, and 8. A significant decline in hemoglobin (Hb) concentration and hematocrit (Hct) was observed at week 5 when all data were examined without regard for iron intake; these red cell indices returned to pre-exercise levels by week 8. Reduction of mean cell hemoglobin concentration (MCHC) indicated that the midpoint decline was not caused by simple hemodilution during exercise. Serum ferritin (SF) concentration changed in parallel with Hb and Hct. Although the midpoint decline in SF was not statistically significant, it ruled out the possibility that turnover of red cell iron was directed to storage. Lowered MCHC and SF suggested lower availability of iron during the synthesis of a new generation of red cells. Few iron treatment effects of magnitude were observed. Iron did not prevent the midpoint decline in Hb concentration. Iron intake did not affect SF, serum iron, transferrin saturation, or final Hb, and Hct. Dietary iron availability thus does not appear to play a role in the phenomenon of “sports anemia”. Temporary alteration of priorities for iron needs during exercise, perhaps for muscle myoglobin, may be responsible for this transitory “anemia”.

Adaptations of lactate metabolism in iron-deficient rats.

Abstract Effects of dietary iron deficiency on lactate metabolism were studied in weanling female rats. Following an iron-deficient diet for 5 weeks, mean hemoglobin concentration was lowered to 6.4 g/dl relative to 12.2 in the control group. Mean plasma iron levels were 58 and 162 μg/dl, respectively. Significantly elevated resting lactate levels were observed in whole blood and plasma from iron-deficient anemic (relative to control) rats. Total activity of lactate dehydrogenase (LDH) was elevated in soleus and gastrocnemius muscles in response to iron deficiency from 269 ± 51 to 364 ± 60 (Mean ± SD) and from 265 ± 65 to 372 ± 61 IU·10−3·g–1, respectively. The LDH activity in heart was lowered from 700 ± 61 to 593 ± 45 IU·10−3·g–1. The M3H and M2H2 isozymes in soleus were increased from 12.7 ± 2.8 to 20.4 ± 5.8% and from 19.4 ± 6.1 to 28.2 ± 3.6%, respectively. Similar increase was observed in M2H2 and MH3 in gastrocnemius from 9.8 ± 0.9 to 14.8 ± 2.0% and from 17.4 ± 2.0 to 20.5 ± 2.3%, respectively. The H4 isozyme was significantly reduced in soleus, gastrocnemius, and plantaris muscles from 27.7 ± 4.7 to 12.4 ± 4.4, from 15.8 ± 1.9 to 7.2 ± 2.9, and from 10.5 ± 2.9 to 3.9 ± 2.1%, respectively. It was suggested that iron-deficiency anemia induces an elevation of lactate production following an increase in total LDH activity and change in LDH isozyme patterns.

Complex formation, polymerization, and autoreduction in the ferric fructose system

Abstract The solution chemistry of ferric fructose has been examined at various hydroxide-to-iron ratios using several physical techniques. The reactivity of ferric fructose complexes with transferrin, the serum iron binding protein, has provided a quantitative measure of polymeric fractions and insight into the rate and mode of breakdown of the polymers. A hydroxy-iron polymer is formed maximally (90%) in the neutral pH region corresponding to 3 OH/Fe, while mononuclear ferric fructose complexes predominate both in acidic (probably as FeFru(-H+)2+, and basic (probably as FeFru(-4H+)− solutions. The chemistry is similar to that of the ferric gluconate system, except that the carboxylate group of gluconate makes it more effective at breaking down the hydrolytic iron polymer. Both ligands form octahedral mononuclear complexes, and gluconate exhibits higher ligand field strength than fructose. The hydrolytic ferric fructose polymer shows the same spectrum as the ferric nitrate and ferric citrate polymers but is much smaller (mw

Resolution of ascorbic, dehydroascorbic, and diketogulonic acids by anion-exchange column chromatography

To investigate the chelating properties of ascorbate and its oxidation products and hydrolytic derivatives, we required a rapid qualitative and quantitative analysis of mixtures of ascorbic, dehydroascorbic, and diketogulonic acid+ 2. Overlapping specificities in the chemical assays 3-b for the individual components made prior fractionation of the mixtures necessary. We therefore developed a chromatographic system for unambiguously separating mixtures on a column of anion-exchange resin using continuous elution with a single eluent. The qualitative specificity of this system takes advantage of differentials in acidity to resolve the components; the separated components are then assayed quantitatively by calorimetric determination of their z,+dinitrophenylosazones.

Mitochondrial NADH dehydrogenase in iron‐deficient and iron‐repleted rat muscle: an EPR and work performance study

Summary. Iron may affect both respiratory O2 transport and mitochondrial electron transport in the performance of muscle work. This study was designed to elucidate the molecular defect iron‐deficient work performance by identifying heretofore unmeasurable mitochondrial enzymes that are diminished by iron deficiency and may be restored by iron repletion. Female rats were made iron‐deficienty by dietary control and were repleted by oral iron. Iron deficiency reduced physical work capacity (treadmill running time), haemoglobin (Hb), and mitochondrial ironsulphur (Fe‐S) centres in heart and skeletal muscles; mitochondrial number was unaffected. Oral iron supplementation restored work capacity and Hb within 4 d to normal or near‐normal levels, but in general Fe‐S centrres of mitochondria due to NADH dehydrogenase remained at iron‐deficient levels. Subnormal concentrations of mitochondrial iron‐dependent NADH dehydrogenase in muscle are not by themselves rate‐limiting in work performance.

Rapid Induction of Ferritin in Laboratory Animals Prior to Its Isolation

Abstract Oral ingestion, by guinea pigs, of concentrated solutions of soluble hydroxy-iron(III) polymers rapidly induces a 7-fold increase of liver ferritin. Young guinea pigs imbibing 0.1 M iron for 2 weeks produced a significantly greater amount of ferritin than animals injected with an iron-dextran complex at 1.0 mmole Fe/kg body weight. Conventional methods for the isolation of tissue ferritin are more efficient and provide higher yields from such iron-replete animals. Dose/response curves are presented for the mouse to illustrate the kinetics of liver iron assimilation at various levels of oral iron supplementation.