A study of the aggregation of human red blood cells induced by picric acid

The effect of picric acid on the aggregation of human erythrocytes was studied. It was shown that the addition of picric acid to a suspension of washed erythrocytes leads to a decrease in pH of medium to 1.5-2 and the formation of echinocytes. Stirring the suspension of echinocytes at low pH values results in a strong aggregation of cells. Increasing the pH value to 7.4 leads to a desaggregation of echinocytes. It was found that picric acid does not induce the aggregation of cells fixed by glutaraldehyde. A substantial decrease in the aggegation of spheric erythrocytes obtained after heating the cells at 50 degrees C was observed.     

Cytochemical localization of phosphatase in differentiating secondary vascular cells

Summary Phosphatase activity was studied in the cambium and differentiating vascular cells of beech by using a modified Washstein and Meisel method. After fixation in glutaraldehyde or crotonaldehyde and incubation in a medium containing ATP and lead nitrate at pH 7.2, a deposit of electron-opaque granules was found in the nucleoli, nucleoplasm, nuclear envelope, endoplasmic reticulum, plastids, mitochondria, and at the plasmalemma. Although located at these different sites, the distribution varied both inter- and intra-cellularly. This is thought to be a true reflection of the variation in activity between closely adjacent cells in this part of the stem.Some reaction was obtained when ADP replaced ATP in the reaction mixture, but there was no reaction at all when both ATP and ADP were omitted. Fixation in hydroxyadipaldehyde, or incubation at pH 6.4 both produced very little reaction.     

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not. This revised version was published online in November 2006 with corrections to the Cover Date.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC (S-nitroso-N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid approximately 90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (approximately 50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.     

Electrophoretic detection of reversible chlorpromazine · HCl binding at the human erythrocyte surface

The binding of chlorpromazine · HCl at the human erythrocyte surface has been detected through its effect on cellular electrophoretic mobility. Incubation of erythrocytes (approx. 5 · 10⁶/ml) in 23 μM chlorpromazine · HCl resulted in a reduction of negative electrophoretic mobility from the control value of $\text{?1.11 ± 0.01}$ (μm · s^?1)/(V · cm^?1) to $\text{?1.00 ± 0.02}$ (μm · s^?1)/(V · cm^?1) (pH 7.2, ionic strength 0.155). This mobility change was completely reversed when chlorpromazine · HCl was removed by centrifugal washing. Increasing the drug concentration to 70μM did not affect the mobility change, indicating saturation of the electrophoretically detectable drug binding sites over chlorpromazine · HCl concentration range studied here. The effect of the 23 μM chlorpromazine · HCl on electrophoretic mobility was also measured in isotonic media of reduced ionic strength. The drug-induced reduction in negative surface charge density was found to be independent of ionic strength over the range 0.155 (Debye length, 0.8 nm) to 0.00310 (Debye length, 5.7 nm).Fixation of erythrocytes with glutaraldehyde affected neither the normal electrophoretic mobility of discocytes nor the reduced electrophoretic mobility of chlorpromazine · HCl-induced stomatocytes. When these stomatocytes were first fixed with glutaraldehyde, then washed free of chlorpromazine · HCl, they retained the stomatocyte form while regaining a normal control electrophoretic mobility. Conversely, when discocytes fixed in that form were treated with chlorpromazine · HCl, they showed the same mobility change as did fixed or unfixed stomatocytes. The drug-induced mobility change is therefore independent of the shape change, but reflects a contribution to cellular surface charge density from the membrane-bound chlorpromazine · HCl molecules. From the charge reduction, it is estimated that about 10⁶ chlorpromazine · HCl molecules are bound at the electrokinetic cell surface and occupy approximately 0.4% of the total surface area.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Effect of acidosis on the membrane potential of intact guinea pig aortic endothelial cells

The effects of a decrease in the extracellular pH from 7.4 to 6.9 on the membrane potential (MP) of intact non-stimulated guinea pig aortic endothelial cells and their ATP-induced electrical responses were studied using a whole-cell mode of the patch-clamp technique. Superfusion of the strip with CO₂-−HCO ₃ ⁻ -buffered acidic solution evoked endothelial depolarization of 6.1±1.0 mV. In Ca²⁺-free medium, after the MP had been stabilized at a depolarized value, there was no shift in the MP due to extracellular acidification to pH 6.9. In the case of using tris-buffered solution, the same drop in the extracellular pH in Ca²⁺-containing medium induced no change in the endothelial MP. Subsequent superfusion with CO₂−HCO ₃ ⁻ -buffered solution with pH 6.9 evoked endothelial depolarization of 7.3±1.5 mV. Changing from tris-buffered to CO₂−HCO ₃ ⁻ -buffered solution at a constant buffer pH 7.4 also induced endothelial depolarization, suggesting that intracellular pH is a possible factor that modulates leak Ca entry. The duration of ATP-induced endothelial hyperpolarization at pH 6.9 significantly dropped (76±5 sec, on average) compared with that at pH 7.4 (121±14 sec). It is concluded that modulatory effect of acidosis on the MP of endothelial cells and their ATP-induced electrical responses are caused by inhibition of leak and ATP-stimulated calcium entry into these cells.     

Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy

We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.     

The interaction of albumin and concanavalin A with normal and sickle human erythrocytes.

The interaction of human albumin and concanavalin A with normal and sickle human red blood cells previously washed in phosphate buffer at pH = 7.4 was studied by titration calorimetry. The amount of albumin bound to normal cells was (6.8 ± 2.2) × 10⁵ molecules/cell. An equilibrium constant of 5 × 10¹⁰ and an enthalpy change of ?(280 ± 90) kcal/mol albumin was determined for albumin interaction with normal cells. The amount of albumin bound to sickle cells was (12.4 ± 1.0) × 10⁵ molecules/cell and the enthalpy change for albumin interaction with sickle cells was ?(390 ± 140) kcal/mol. Normal cells bound (5.7 ± 2.4) × 10⁵ concanavalin A molecules/cell with an enthalpy change of ?(840 ± 200) kcal/mol concanavalin. All experiments were conducted at 25°C.     

Possibility of the long-term fixation of the cells of a HeLa culture without disturbing their ultrastructure

It is shown that the composition of fixatives (2.5% glutaraldehyde in 0.1 M phosphate buffer with pH 7.2-7.4, and the mixture of 2.5% glutaraldehyde with 2% paraformaldehyde in 0.1 M phosphate buffer with pH 7.2-74) and duration of fixation (30 minutes, 24 hours, 7 days, and 30 days) under the room temperature exerts no influence on preservation of HeLa cultured cells. The ultrastructure of all the organelles of these cells is similar in any cases examined. All the membrane structures are well preserved; no condensation of chromatin is observed; the widths of the canals of endoplasmic reticulum, and of the intracristal and lateral spaces of mitochondria are invariable. Polysomes are present in the cytoplasm throughout the period of fixation.     

Gravity sedimentation analysis of human blood leukocytes

A new method of sedimentation analysis of human blood leukocytes is described. Platelets, lymphocytes, monocytes, and polymorphonuclear cells isolated from normal human peripheral blood have been analyzed alone and in mixture by gravity sedimentation employing a computerized scanning instrument. All four classes could be clearly resolved from each other exhibiting sedimentation velocities of 0.6 ± 0.00, 1.04 ± 0.11, 1.27 ± 0.15 and 1.89 ± 0.21 · 10^?4 cm/s, respectively, at 37°C in a 2.5–6.25% Ficoll gradient in Medium 199. Less than 10⁶ cells can be used for analysis. Possible applications of the method are discussed.     

A simple two-dye basic stain facilitating recognition of mitosis in plastic embedded tissue sections

Semithin sections of buccal and palatal mucosa fixed in 2.5% glutaraldehyde followed by 1% osmium and embedded in Durcupan (an araldite-based resin) were stained with 2% malachite green in 50% ethanol at 80 C and poststained in 0.05% crystal violet in Sorensen's phosphate buffer (pH 6.4) at 45 C. Nuclear envelopes and chromatin stain vivid purple in contrast to the surrounding green cytoplasm and cell borders. Chromosomes of dividing cells stain bluish violet. Nucleoli, depending on their level in the epithelium, stain differing shades of greenish blue. The distinct and differential staining of each of these components facilitates recognition of mitoses in oral epithelium, where the small size and crowding of cells in the proliferative compartment renders more conventional stains for plastic sections inadequate.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.     

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.     

The effects of different pretreatment conditions and fixation regimes on serotonin immunoreactivity: a quantitative light microscopic study

An investigation was designed to evaluate the effects of three different fixation regimes on the retention of serotonin-like immunoreactivity in rat midbrain tissue sections. The effects of pretreatment with pargyline-HCl and l-tryptophan on the volume fraction of serotonin-like immunoreactive processes were also examined. Rat brain tissue was fixed with 4% paraformaldehyde (Pf), 4% paraformaldehyde-0.2% picric acid-0.05% glutaraldehyde (Pf-Pa-G), or 4% paraformaldehyde-0.2% glutaraldehyde (Pf-G). Tissue was subsequently processed for immunohistochemistry using a modified peroxidase-antiperoxidase technique and quantified at the light microscopic level by point counting. Fixation with Pf resulted in higher volume fraction determinations of axonal serotonin immunoreactivity than did fixation with Pf-Pa-G or Pf-G. These results provide quantitative data which indicate that even low levels of glutaraldehyde in the fixative significantly decrease serotonin immunoreactivity. Pretreatment with pargyline and tryptophan increased the amount of serotonin immunoreactivity in tissue fixed with Pf-G but not in tissue fixed with Pf. Pretreatment with pargyline and tryptophan is thus recommended when using glutaraldehyde in the fixation process to assure adequate serotonin immunoreactivity. Pretreatment in conjunction with glutaraldehyde fixation, however, appears to cause differential increases in serotonin-like immunoreactivity within brain nuclei that may compromise the interpretation of results.     

Fixation and embedding variables in the immuno-electron microscopic study of rat heymann nephritis

Summary Fixation and embedding variables were compared in immuno-electron microscopic localization of rat IgG in an autologous immune complextype nephritis. Specimens from kidney cortex were fixed for 3, 6 or 9 h in the following fixatives made in 0.1 M phosphate buffer at pH 7.44% paraformaldehyde, 2.5% glutaraldehyde, periodate-lysine-paraformaldehyde or modified Karnovsky's fixative. Localization of IgG was performed on tissue sections cut with a tissue chopper, cryostat or sliding microtome, using agarose, Ames O.C.T. Compound or polyethylene glycol respectively as cutting matrixes. The sections were incubated in peroxidase-labelled antirat IgG antiserum (diluted 120 with phosphate-buffered saline) for 60 h. Peroxidase activity was then revealed and the sections embedded in Epon. Exact localization of IgG throughout the sections and good ultrastructure were achieved when paraformaldehyde and agarose were used. Periodate-lysine-paraformaldehyde proved almost as useful as paraformaldehyde in connection with agarose in respect of peroxidase reaction and ultrastructure. Fixatives containing glutaraldehyde gave a mostly weak and unevenly distributed peroxidase reaction product. In the cryostat sections breaking of the tissue structure could not be avoided. When polyethylene glycol was used as cutting matrix no peroxidase reaction was achieved.     

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering,sedimentation, and circular dichroism studies on the holo- and apoproteins

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 Å) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 ų), surface (11,840 ± 120 vs 13,470 ± 140 Å), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H₂O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.