Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.     

Aqueous two-phase partitioning sample preparation prior to liquid chromatography of hydrophilic drugs in blood

The technique of aqueous two-phase partitioning is investigated as a sample preparation procedure prior to LC determination of drugs in blood. In the extraction 500 microl of whole blood is added to 2.00 g of PEG 300 and 18.0 g of phosphate buffer. The phases are mixed by stirring and allowed to separate before the clear lower salt phase is drained out using a specially designed glass adapter. After filtration 20 microl is injected into the LC system. When validated for the X-ray contrast medium iohexol R.S.D. was 2.0% and 1.2% at 10 microg/ml and 100 microg/ml of iohexol in blood, respectively.     

Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol)-lipid

Surface-grafted polymers, such as poly(ethylene glycol) (PEG), provide an effective steric barrier against surface-surface and surface-macromolecule interactions. In the present work, we have studied the exchange of monooleoylphosphatidylcholine (MOPC) with vesicle membranes containing 750 mol wt surface-grafted PEG (incorporated as PEG-lipid) from 0 to 20 mol % and have analyzed the experimental results in terms of thermodynamic and stationary equilibrium models. Micropipette manipulation was used to expose a single lipid vesicle to a flow of MOPC solution (0.025 microM to 500 microM). MOPC uptake was measured by a direct measure of the vesicle area change. The presence of PEG(750) lipid in the vesicle membrane inhibited the partitioning of MOPC micelles (and to some extent microaggregates) into the membrane, while even up to 20 mol % PEG-lipid, it did not affect the exchange of MOPC monomers both into and out of the membrane. The experimental data and theoretical models show that grafted PEG acts as a very effective molecular scale "filter" and prevents micelle-membrane contact, substantially decreasing the apparent rate and amount of MOPC taken up by the membrane, thereby stabilizing the membrane in a solution of MOPC that would otherwise dissolve it.     

Temperature dependence of polypeptide partitioning between water and phospholipid bilayers

Various thermodynamic forces (e.g., the hydrophobic effect, electrostatic interactions, peptide immobilization, peptide conformational changes, "bilayer effects," and van der Waals dispersion forces) can participate in the transfer of polypeptides from aqueous solution into lipid bilayers. To investigate the contributions of these forces to peptide-membrane thermodynamics, we have studied the temperature dependence of the water-bilayer partitioning of 4 polypeptides derived from the first 25 amino acid residues in the presequence of subunit IV of yeast cytochrome c oxidase (Cox IVp) using electron paramagnetic resonance spectroscopy. The partitioning of the Cox IVp peptides into phospholipid bilayers increases as the temperature is increased from 3 to 40 degrees C. The contribution of bilayer surface expansion to the temperature-dependent partitioning is estimated to be relatively small and to contribute minimally to the increased bilayer binding of the peptides with increasing temperature. Thermodynamic analysis of the data shows that the transfer of the peptides from water into bilayers at 298 K is driven by the entropic term (-T delta Str) with values ranging from -6.7 to -10 kcal mol-1, opposed by the enthalpic term (delta Htr) by approximately 4 kcal mol-1, and accompanied by a change in heat capacity (delta Cp) ranging from -117 to -208 cal K-1 mol-1. Our results indicate that while a variety of forces do, in fact, contribute to the transfer free energies (delta Gtr), the major driving force for the water-to-bilayer transfer is the hydrophobic effect.     

Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity

BACKGROUND: Erythropoietin (EPO), the hormone involved in red blood cell production, activates its receptor by binding to the receptor's extracellular domain and presumably dimerizing two receptor monomers to initiate signal transduction. EPO-mimetic peptides, such as EMP1, also bind and activate the receptor by dimerization. These mimetic peptides are not as potent as EPO, however. The crystal structure of the EPO receptor (EBP) bound to EMP1 reveals the formation of a complex consisting of two peptides bound to two receptors, so we sought to improve the biological activity of EPO-mimetic peptides by constructing covalent dimers of EMP1 and other peptide mimetics linked by polyethylene glycol (PEG). RESULTS: The potency of the PEG-dimerized EPO peptide mimetics both in vitro and in vivo was improved up to 1,000-fold compared to the corresponding peptide monomers. The dimers were constructed using peptide monomers which have only one reactive amine per molecule, allowing us to conclude that the increase in potency can be attributed to a structure in which two peptides are linked through their respective amino termini to the difunctional PEG molecule. In addition, an inactive peptide was converted into a weak agonist by PEG-induced dimerization. CONCLUSIONS: The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.     

Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte

A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.     

Chloroaluminum sulfonated phthalocyanine partitioning in normal and intimal hyperplastic artery in the rat. Implications for photodynamic therapy

Photodynamic therapy, the light activation of photosensitizers into cytotoxic mediators, has been a successful treatment for experimental intimal hyperplasia (IH). To understand the basis of the photosensitizer chloroaluminum sulfonated phthalocyanine (CASPc)-mediated photoinhibition of intimal hyperplasia in the rat common carotid artery model, we studied photosensitizer partitioning in hyperplastic as compared to normal arterial tissue. Serum clearance of CASPc is exponential with, a half-life of 300 minutes. Laser-induced fluorescence and spectrofluorimetric analyses of artery tissue demonstrated an approximately 60% lower uptake and retention of CASPc by normal arterial tissue as compared to arteries with IH; the differences become more pronounced at 24 h. Fluorescent microscopy of arterial tissue demonstrated increased uptake of the CASPc by the artery with IH. However, by 24 h it is primarily the IH tissue that has retained the CASPc, with clearance of the dye from the media of normal or hyperplastic arteries. These data demonstrate that IH, like neoplastic tissue, has an increased accumulation of CASPc compared to normal artery. The preferential partitioning into hyperplastic tissue has implications for therapeutic targeting of this cellular population with photodynamic therapy.     

Folding of beta-sheet membrane proteins: a hydrophobic hexapeptide model

Beta-sheets, in the form of the beta-barrel folding motif, are found in several constitutive membrane proteins (porins) and in several microbial toxins that assemble on membranes to form oligomeric transmembrane channels. We report here a first step towards understanding the principles of beta-sheet formation in membranes. In particular, we describe the properties of a simple hydrophobic hexapeptide, acetyl-Trp-Leu5 (AcWL5), that assembles cooperatively into beta-sheet aggregates upon partitioning into lipid bilayer membranes from the aqueous phase where the peptide is strictly monomeric and random coil. The aggregates, containing 10 to 20 monomers, undergo a relatively sharp and reversible thermal unfolding at approximately 60 degreesC. No pores are formed by the aggregates, but they do induce graded leakage of vesicle contents at very high peptide to lipid ratios. Because beta-sheet structure is not observed when the peptide is dissolved in n-octanol, trifluoroethanol or sodium dodecyl sulfate micelles, aggregation into beta-sheets appears to be an exclusive property of the peptide in the bilayer membrane interface. This is an expected consequence of the hypothesis that a reduction in the free energy of partitioning of peptide bonds caused by hydrogen bonding drives secondary structure formation in membrane interfaces. But, other features of interfacial partitioning, such as side-chain interactions and reduction of dimensionality, must also contribute. We estimate from our partitioning data that the free energy reduction per residue for aggregation is about 0.5 kcal mol-1. Although modest, its aggregate effect on the free energy of assembling beta-sheet proteins can be huge. This surprising finding, that a simple hydrophobic hexapeptide readily assembles into oligomeric beta-sheets in membranes, reveals the potent ability of membranes to promote secondary structure in peptides, and shows that the formation of beta-sheets in membranes is more facile than expected. Furthermore, it provides a basis for understanding the observation that membranes promote self-association of beta-amyloid peptides. AcWL5 and related peptides thus provide a good starting point for designing peptide models for exploring the principles of beta-sheet formation in membranes.     

Buccal absorption of enalapril and lisinopril

OBJECTIVE: The buccal absorption of captopril does not exhibit the classical pH/partition hypothesis, suggesting that mechanisms other than passive diffusion are involved in its absorption; animal studies have suggested that a peptide carrier-mediated transport system may be responsible for its absorption. The present study evaluated the effects of pH on octanol partitioning, and on the buccal absorption of enalapril and lisinopril, using in vitro techniques and buccal partitioning in human volunteer subjects. METHODS: The partitioning of enalapril and lisinopril into n-octanol was examined over the pH range of 3 9 at room temperature. RESULTS: Enalapril exhibited maximal partitioning into the organic phase at pH 4 5; minimal partitioning was recorded at pH values 8 and 9. The partitioning of lisinopril into n-octanol was found to be maximal at pH 9 and minimal at pH 3. Using the buccal absorption technique, the partitioning of enalapril and lisinopril (0.5 mg), was examined in six healthy male volunteers from buffered solutions (pH 3, 4, 5, 6, 7, 8 and 9). In the case of enalapril, lowest buccal partitioning occurred at pH 3, 8 and 9, while maximal partitioning occurred at pH 5; absorption of lisinopril was not extensive at any pH, but was greatest at pH 6. These results, in addition to the n-octanol partition coefficients, indicated that enalapril obeyed the normal lipid partition hypothesis with respect to buccal absorption. The buccal absorption of lisinopril also obeyed the lipid partition hypothesis over the pH range 3-7. These findings are in direct contrast to those for captopril. The buccal partitioning experiments were repeated at the maximal pH for absorption for each angiotensin converting enzyme (ACE) inhibitor, but with the addition of cephradine (0.05 mmol x l(-1)). CONCLUSION: The data indicated that the presence of this peptide transport inhibitor had no effect on the buccal absorption of enalapril (0.06 mmol x l(-1)) and lisinopril (0.057 mmol x l(-1)), which suggests that both drugs do not share a common buccal absorption pathway with cephradine.     

Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin

Membranes have a potent ability to promote secondary structure formation in a wide range of membrane-active peptides, believed to be due to a reduction through hydrogen bonding of the energetic cost of partitioning peptide bonds. This process is of fundamental importance for understanding the mechanism of action of toxins and antimicrobial peptides and the stability of membrane proteins. A classic example of membrane-induced folding is the bee-venom peptide melittin that is largely unstructured when free in solution, but strongly adopts an amphipathic alpha-helical conformation when partitioned into membranes. We have determined the energetics of melittin helix formation through measurements of the partitioning free energies and the helicities of native melittin and of a diastereomeric analog with four d-amino acids (d4,l-melittin). Because D4,l-melittin has little secondary structure in either the free or bound forms, it serves as a model for the experimentally inaccessible unfolded bound form of native melittin. The partitioning of native melittin into large unilamellar phosphocholine vesicles is 5.0(+/-0.7) kcal mol-1 more favorable than the partitioning of d4,l-melittin (1 cal=4.186 J). Differences in the circular dichroism spectra of the two forms of melittin indicate that bound native melittin is more helical than bound d4, l-melittin by about 12 residues. These findings disclose that the free energy reduction per residue accompanying the folding of melittin in membrane interfaces is about 0.4 kcal mol-1, consistent with the hypothesis that hydrogen bonding reduces the high cost of partitioning peptide bonds. A value of 0.6 kcal mol-1 per residue has been observed for beta-sheet formation by a hexapeptide model system. These two values provide a useful rule of thumb for estimating the energetic consequences of membrane-induced secondary structure formation.     

聚乙二醇对锂电铜箔组织性能的影响

利用直流电沉积技术在纯钛阴极上制备电解铜箔,在含有光亮剂和整平剂的电解液中加入0~6 mg/L聚乙二醇（PEG）,对电解铜箔表面状态和力学性能均产生影响。随着PEG浓度增加,铜箔抗拉强度轻微增加;过量的PEG则导致铜箔表面粗糙度增大。通过扫描电子显微镜（SEM）观察铜箔表面形貌发现：加入0~4 mg/L PEG可制得表面较为平整的电解铜箔,过量的PEG会使得铜箔表面出现球状突起。     

The design, synthesis, and initial evaluation of benzophenone-containing peptides as potential photoaffinity labels of oligosaccharyltransferase

The benzophenone photophore was incorporated into protected tripeptides and tetrapeptides as photoactivatable probes to study the multimeric enzyme oligosaccharyltransferase (OST). These peptides contain the -Asn-X-Thr- sequon which is required for OST-catalyzed N-glycosylation. Two tripeptides, Bz-Asn-Bpa-Thr-NH2 (3b) and Bz-Asn-Lys[N epsilon-(4-Bz)Bz]-Thr-NH2 (4b), were found to be good OST substrates. They were competitive inhibitors versus standard peptide substrate [14C]Bz-Asn-Leu-Thr-NH2 and their Ki values were determined to be 41 +/- 6 microM and 21 +/- 6 microM, respectively, using synthetic (GlcNAc)2-PP-dolichol.     

Substrate specificity of mammalian prenyl protein-specific endoprotease activity

We have previously identified proteolytic activity in rat liver microsomes that cleaves an intact tripeptide, VIS, from S-farnesylated-CVIS tetrapeptide. This enzymatic activity, termed prenyl protein-specific endoprotease (PPEP) activity, has been solubilized in CHAPS and purified 5-fold. To probe the peptide recognition features of PPEP activity, 64 tripeptides [N-acetyl-C(S-farnesyl)a1a2] were prepared and tested as competitive inhibitors of PPEP activity-catalyzed hydrolysis of N-acetyl-C(S-farnesyl)VI[3H]S. It was found that PPEP activity prefers large hydrophobic residues in the a1 and a2 positions. A subset of N-acetyl-C(S-farnesyl)a1a2 peptides were prepared in radiolabeled form, and it was found that PPEP activity preferences for these substrates correlated well in most cases with the inhibition data. The exception is that R in the a1 position does not prevent binding of peptide to PPEP activity, but such peptides are poor substrates. The anionic residue D in the a2 position is not tolerated by PPEP activity. Five farnesylated radiolabeled tetrapeptides, Ac-C(F)FM[3H]L, Ac-C(F)LI[3H]L, Ac-C(F)LL[3H]L, Ac-C(F)LM[3H]L, and Ac-C(F)VI[3H]L were prepared, and PPEP activity kinetic studies revealed that they are good substrates and show comparable KM values (2.2-13.5 microM). Ac-C(F)RL[3H]S is a poor substrate. The reported peptide binding preferences of PPEP activity should be useful in designing compounds that block the C-terminal proteolysis of prenylated proteins. Nonprenylated peptides do not bind to PPEP activity, and replacement of the farnesyl group with ann-pentadecyl group modestly reduces binding. Peptide-membrane partitioning studies were used together with theoretical arguments to fully understand the substrate specificity of PPEP activity toward these compounds.     

Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

Role of water in protein kinase C catalysis and its binding to membranes

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.     

Effect of Oil on Polychlorinated Biphenyl Phase Partitioning during Land Biotreatment of Impacted Sediment

This study evaluated the partitioning of polychlorinated biphenyls (PCBs) during long-term, passive, land biotreatment of PCB-impacted industrial lagoon sediments. Over six years under field conditions, two land treatment units (LTUs) experienced 40% total PCB reductions from initial concentrations of 8–10?mg/kg. A third LTU with 113?mg/kg initial total PCBs showed little reduction over five years. In each unit throughout the study, oil concentrations declined at a rate greater than that for PCBs. Measured aqueous equilibrium concentrations for the PCB-impacted sediments were typically an order of magnitude or more smaller than values estimated using correlations based on total organic matter partitioning. Measured aqueous PCB concentrations agreed with predictions based on equilibration with a PCB-containing oil phase, best modeled by Raoult’s law. It was postulated that, as a consequence of PCB oil-phase partitioning, biotreatment would lead to higher PCB concentrations in the oily matter and thus increased PCB partitioning to the aqueous phase if the degradation of oily matter proceeded faster compared to PCBs. Such was the case in this study, wherein low-level aqueous phase PCB concentrations of tetrachloro PCBs increased several fold over the years as oily matter was degraded. The contribution of oil to PCB partitioning needs to be incorporated in the assessment of risk and treatability goals for land biotreatment of contaminated sediments from industrial sites.     

Effect of percutaneous endoscopic gastrostomy on gastric emptying in clinically normal cats

OBJECTIVE: To assess the effect of percutaneous endoscopic gastrostomy (PEG) tube placement on gastric emptying in clinically normal cats. ANIMALS: 8 healthy adult 3- to 5-year-old cats. PROCEDURE: Cats were accommodated to the diet for 2 weeks prior to scintigraphy. Caloric needs were divided into 3 feedings/d. Food was withheld for 24 hours after tube placement, then was fed as a third of the caloric needs on day 1, two-thirds on day 2, and full caloric requirements thereafter. Gastric emptying was measured via nuclear scintigraphy. Labeled meals contained 111 MBq (3 mCi) of 99mTc-labeled disofenin. Sixty-second ventral scintigraphic images were acquired immediately, every 20 minutes for the first hour, then every 30 minutes for 4 hours after feeding. Each cat was evaluated 3 times prior to PEG tube placement. Cats were anesthetized, and 16-F mushroom-tipped Pezzar gastrostomy tubes were placed, using a video endoscope. Scintigraphy was repeated on days 1, 4, 7, 11, 14, and 21 after PEG tube placement. RESULTS: Gastric emptying was faster with a PEG tube in place. Percentage of retained gastric activity was significantly lower after PEG for 150, 180, 210, and 240 minutes versus time before PEG tube placement. CONCLUSION: Placement of a PEG tube does not delay gastric emptying in clinically normal cats. CLINICAL RELEVANCE: Gastric retention of food, vomiting, and aspiration pneumonia after PEG tube placement may not be related to delayed gastric emptying.     

Prediction of beta-turns

A residue-coupled model is proposed to predict the beta-turns in proteins. The rates of correct prediction for the 455 beta-turn tetrapeptides and 3807 non-beta-turn tetrapeptides in the training database are 94.7 and 81.3%, respectively. The rates of correct prediction for the 110 beta-turn tetrapeptides and 30,229 non-beta-turn tetrapeptides in the testing database are 80.0 and 80.2%, respectively. Compared with the rates of correct prediction based on the residue-independent model reported previously, the quality of prediction is significantly improved by the new model, implying that the residue-coupled effect along a polypeptide chain is important for the formation of reversal turns, such as beta-turns, during the process of protein folding.     

Influence of organic modifier concentration on plate number in micellar electrokinetic chromatography. 1. 2-propanol

This paper establishes a physicochemical basis for the efficiency losses in micellar electrokinetic chromatography in buffers containing sodium dodecyl sulfate (SDS) and 2-propanol (2PN). Weakly, intermediately, and strongly retained analytes were separated in phosphate/borate buffers containing 50 mM SDS and from 0 to 10% 2PN by volume. Their plate numbers N generally agreed well with predictions of a theory for N based on longitudinal diffusion and instrumental contributions to dispersion. The N's of weakly and intermediately retained analytes were not affected strongly by 2PN over this concentration range, because their diffusion coefficients varied inversely with buffer viscosity and their retention times largely varied directly with viscosity. These combined effects on dispersion almost canceled. However, the N's of strongly retained analytes decreased with increasing 2PN, because their diffusion coefficients varied inversely with viscosity but their retention times increased more rapidly than did viscosity. These combined effects on dispersion did not cancel. These differences occurred because 2PN penetrated the micelles, caused bound counterions to be released, and increased the micellar charge and electrophoretic mobility. As 2PN concentration increased, the micelles electrophoresced increasingly rapidly against the electroosmotic flow. Consequently, strongly retained compounds required increasingly long times to elute.