Dynamics of amino acid residues in globular proteins

The dynamic differential equation model developed and tested for bovine pancreatic trypsin inhibitor and tuna ferrocytochrome c in Ponnuswamy, P.K. & Bhaskaran, R. (Int. J. Peptide Protein Res. 24 , 168–179, 1984) is extended for 17 more protein crystals in this work. Average displacements are computed for 20 amino acid residues observed in 19 proteins. Detailed information on the dynamic behaviour of the individual proteins and individual residues is presented. The effect of atomic packing on the fluctuations of the amino acid residues in α-chymotrypsin is illustrated. A number of general points on the dynamic characteristics of globular protein molecules are presented.     

Formulation and stability of a novel artificial human sweat under conditions of storage and use

A limitation of most artificial sweat formulations used for in vitro assessment of chemical release from materials in contact with skin have little biological relevance to human sweat. The purposes of this paper are to provide guidance for preparation of a novel artificial sweat with chemical constituents at concentrations that match human sweat and to characterize chemical stability. The artificial sweat was characterized under conditions of use (with and without sebum at 36 °C) and storage (without sebum at −4, 4, and 23 °C) over 28 days by gas chromatography–mass spectroscopy, high-performance liquid chromatography, enzymatic assay kits, and ion-selective electrodes. Seven indicator constituents were tracked: sodium, chloride, glucose, lactic acid, urea, pantothenic acid, and alanine. With or without sebum at 36 °C, the sweat solvent was chemically stable for 14 days. Storage by refrigeration at 4 °C retained the chemical integrity of the solvent longest. Based on these results, the solvent should be used within 14 days of preparation. The artificial sweat model presented herein is most similar to human sweat and has applications as a dissolution solvent, donor solution in diffusion cells, or vehicle for patch testing. This sweat model may aid researchers in understanding potential release and percutaneous absorption of chemicals in contact with human skin surface liquids.     

Positional flexibilities of amino acid residues in globular proteins

The fluctuational amplitudes of the amino acid residues in 19 protein molecules are computed by the use of our differential equation model and are analysed statistically to get new information as to their stability, position dependent nature, distribution and group dynamical behaviour, etc. The symmetric/asymmetric distribution of amino acid residues in the protein molecules is described by introducing a parameter called the “flexibility index” for each of the residue types. Finally, the overall flexible nature of the amino acid residues with respect to their spatial positioning is explained and their relationships with the residue properties are derived.     

Preparation optimisation and storage stability of nanoemulsion-based lutein delivery systems

Abstract

The present work formed lutein-enriched nanoemulsions stabilised by sodium caseinate (SC) using a high-pressure homogenisation process, and the influence of environmental conditions on the physicochemical stability of the nanoemulsion was investigated. The results showed that the droplet diameter of the nanoemulsion was largely dependent on homogenisation conditions. Optimum results were obtained for 1.0% (w/w) SC, 100?MPa pressure, and 7 homogenisation cycles, which produced a nanoemulsion with a mean droplet diameter of 234.01?±?3.40?nm, polydispersity index of 0.123?±?0.028, and zeta potential of ?36.56?±?1.51?mV. The nanoemulsion remained physically stable after a 30 d storage at 4?°C, and the chemical degradation rate of lutein was considerably decreased. Thermal treatment at 60–100?°C had little effect on its physicochemical stability; conversely, pH, ionic strength (NaCl or CaCl₂), concentration treatment, and freeze–thaw cycling had major impacts on the physicochemical stability of nanoemulsion.     

3%必需氨基酸注射液质量与稳定性研究

目的建立3%必需氨基酸注射液含量测定的分析方法并进行稳定性的研究。方法采用L-8900高速氨基酸分析仪测定注射液的含量,进行制剂冷冻加热循环试验、制剂加热与光照试验、制剂的加速稳定性试验。结果建立含量测定方法符合方法学要求,稳定性试验符合要求。结论本文所建立的氨基酸分析方法简便、准确,所制备的3%必需氨基酸注射液稳定性较好。     

氨基酸分析仪法测定丙氨酰-谷氨酰胺的含量及输液配伍的稳定性

目的建立茚三酮柱后衍生化氨基酸分析仪测定丙氨酰-谷氨酰胺含量的方法,以评价丙氨酰-谷氨酰胺注射液与配伍输液的稳定性。方法根据丙氨酰-谷氨酰胺与茚三酮反应后溶液在570nm处有最大吸收的原理,用氨基酸分析仪测定丙氨酰-谷氨酰胺的含量。将丙氨酰-谷氨酰胺注射液以1:5体积比分别与氯化钠注射液、5%葡萄糖注射液、5%葡萄糖氯化钠注射液、10%果糖注射液、乳酸林格液、复方氨基酸注射液以及含多种微量元素的复方氨基酸注射液配伍,室温下,6h内检查性状以及含量的变化。结果丙氨酰-谷氨酰胺在1～20mg·L~(-1)范围内线性关系良好(r=0.997 9),低、中、高3种浓度的回收率分别为94.5%、97.6%和98.7%;RSD分别为0.52%、0.10%和0.06%。丙氨酰-谷氨酰胺注射液分别与上述各组输液配伍后,6h内外观未见明显变化;0～6h复方氨基酸注射液、5%葡萄糖注射液分别与丙氨酰-谷氨酰胺注射液配伍,丙氨酰-谷氨酰胺的含量变化无显著差异(P>0.05);其余各组输液与丙氨酰-谷氨酰胺注射液配伍后,组间差异非常显著(P<0.01)。结论该法操作简单,分离度良好,结果准确可靠,可用于丙氨酰-谷氨酰胺的含量测定。丙氨酰-谷氨酰胺注射液的最佳载体为5%葡萄糖注射液和复方氨基酸注射液。     

Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility

The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized protein products. Five proteins were freeze dried with different amounts of sucrose, and protein aggregation was quantified using Size Exclusion Chromatography. Protein secondary structure was monitored by FTIR. The global mobility was studied using Thermal Activity Monitor (TAM), and fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the protein formulations was measured with a gas pycnometer. The physical stability of the proteins increased monotonically with an increasing content of sucrose over the entire range of compositions studied. Both FTIR structure and structural relaxation time from TAM achieved maxima at about 1:1 mass ratio for most proteins studied. Therefore, protein stabilization by sugar cannot be completely explained by global dynamics and FTIR structure throughout the whole range of compositions. On the other hand, both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the formulations, and thus the local dynamics and free volume correlate well with protein storage stability. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3145–3166, 2009     

A Dual Controlled Gastrointestinal Therapeutic System of Salmon Calcitonin. II. Screening of Process and Formulation Variables

An important aspect of the “Desired State” of manufacturablity as defined by the International Committee of Harmonization is the mechanistic understanding and predictability of dosage forms at the laboratory scale. The accomplishment of that aspect is often preceded by a formulation knowledge and previous history of the project or by screening of the variables to identify the critical ones. Osmotically controlled drug delivery systems provide a means of eliminating the effect of pH, food as well as transit time on drug release. Salmon calcitonin, a hypocalcemic peptide, was formulated as an osmotically controlled bilayered enteric‐coated dosage form with turkey ovomucoid (enzyme inhibitor) and glycyrrhetinic acid (permeation enhancer) along with other excipients. Drug release from the dosage form is generally affected by formulation and process variables. However, the literature information is very limited for the effects of these variables on the release kinetics of peptide drugs from osmotically controlled systems. The objective of this study was to evaluate the factors that influence the release of the drug from bilayered, osmotically controlled tablets coated with a semipermeable membrane of cellulose acetate. A seven‐factor‐12‐run Plackett‐Burman screening technique was employed to evaluate the effects of orifice size, coating level, amounts of sodium chloride, Polyox® N10 and N80 and Carbopol® 934P and 974P on drug release. Response variables was cumulative percent released in 24 hr with constraints on time for 25% and 50% drug release. Factors showing maximum influence on drug release were amounts of Carbopol® 934P and Polyox® N10 in the drug layer, orifice size and coating level showing negative effects with the main effect magnitudes of ? 30.85, ? 10.97, ? 9.61, and ? 9.95, respectively.     

Hydrophobic distribution and spatial arrangement of amino acid residues in membrane proteins

The analysis of known three-dimensional structures of membrane proteins provides an opportunity to understand their structure and stability. In this article we analyse the hydrophobic variation of amino acid residues at various ranges in membrane and aqueous parts of membrane proteins. The numerical indices for several properties of amino acid residues in membrane proteins, such as surrounding hydrophobicity, gain in surrounding hydrophobicity, hydrophobic gain ratio, accessible surface area, preference of amino acid residues in the interior and surface parts, solvent accessible reduction ratio and buriedness, were set up. The relative preference of amino acid residues at various positions of membrane proteins were obtained in a very realistic approach. © Munksgaard 1996.     

Formulation and stability of cefuroxime eye-drops

The recent clinical demand for cefuroxime eye-drops has given rise to practical difficulties because of the short shelf life recommended by the manufacturer of Zinacef injection, the product used to prepare the eye-drops. The purpose of this investigation was to determine the stability of various formulations of cefuroxime sodium eye-drops by high pressure liquid chromatography. The results showed that buffering the solution in the pH range 6.0 to 7.3 has little effect on the shelf life when compared with a simple aqueous solution of 5 per cent cefuroxime, which retains satisfactory potency for 21 days in a refrigerator at 2°C during simulated patient usage. Although stable for only 24 hours at room temperature, negligible loss of potency is observed after storing the eye-drops for 12 months at -30°C. Frozen solutions should be defrosted at room temperature and shaken thoroughly to redisperse the cefuroxime evenly throughout the solution. Thawed solutions are stable in the refrigerator for a further 21 days at 2°C or for 14 days at 8°C. As temperatures up to 8°C are often recorded with domestic refrigerators, a 14 day shelf life is recommended in the refrigerator.     

Partial amino acid sequence of porcine elastase II

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (M_r = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser¹⁹⁵. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Ascorbic acid in thyroidectomized rats. II) Ascorbic acid status of the storage tissues and hepatic biosynthesis of glucuronic acid

Depletion of ascorbic acid from adrenals, brain and epididymis along with loss in weight were noticed in the state of thyroidectomy. This decrease appears to be due to an effect of thyroidectomy on the membrane integrity since the membrane bound sialic acid was found to be significantly lowered in these tissues as a consequence of the elevated activity of sialidase. Thyroidectomy was also found to cause an adverse effect on the activities of hepatic UDP-glucuronyl transferase and beta-glucuronidase with no alteration in UDP-glucose dehydrogenase.     

On the stability of ascorbic acid in emulsified systems for topical and cosmetic use.

Several O/W microemulsions, O/W and W/O emulsions and a W/O/W multiple emulsion were prepared using non-ionic, non-ethoxylated, skin compatible emulsifiers. Ascorbic acid (vitamin C) was added to the emulsified systems and its stability against oxidation was studied at 45.0 degrees C in aerobic conditions and compared with that in aqueous solutions at different pH values. All emulsified systems provided protection to ascorbic acid, as its degradation rate, which increased with increasing pH, was slower in emulsified systems than in aqueous solutions. The highest protection of ascorbic acid was when it was dissolved in the inner aqueous phase of the W/O/W multiple emulsion, both at 45 and at 20 degrees C for long storage. A pseudo first-order mechanism was hypothesised for ascorbic acid degradation in the experimental conditions for as long as abundant dissolved oxygen was present.     

Synthesis of angiotensin II antagonists containing N- and O-methylated and other amino acid residues.

[1-N-Methylisoasparagine,8-isoleucine]- (I), [1-sarcosine,4-N-methyltyrosine,8-isoleucine]- (II), [1-sarcosine,5-N-methylisoleucine,8-isoleucine]- (III), [1-sarcosine,8-N-methylisoleucine]- (IV), [1-sarcosine8k-N-methylisoleucine,8-N-methylisoleucine]- (V), [1-sarcosine,8-O-methylthreonine]- (VI), [1-sarcosine,8-methionine]- (VII), and [1-sarcosine,8-serine]angiotensin II (VIII), synthesized by Merrifield's solid-phase procedure, possess respectively 0.8, 0.3, 0.5, 1.0, 0.0, 0.5, 3.7, and 0.7% pressor activity of angiotensin II (vagotomized, ganglion-blocked rats). They caused an initial rise in blood pressure (30 min of infusion, 250 ng/kg/min in vagotomized, ganglion-blocked rats) of 16.57, 9.80, 22.80, 32.00, 7.00, 15.06, 32.50, and 11.42 mmHg and showed secretory activity (isolated cat adrenal medulla) of 1.0, 0.1, 0.01, 0.1, less than 0.01, 0.1, less than 0.01, and 0.05% of angiotensin II. On isolated organs pA2 values (rabbit aortic strips) of 8.74, 7.44, 7.64, 7.85, 7.89, 8.76, 8.63, and 8.08, and pA2 values (cat adrenal medulla of 8.16, 9.16, 9.31, 8.00, 8.00, 7.00, 9.16, and 9.33 were obtained. Dose ratios (ratio of ED20 of angiotensin II during infusion of the antagonist and before infusion of the antagonist) in vagotomized, ganglion-blocked rats, infused at 250 ng/kg/min, were 33.43, 2.14, 3.26, 2.99, 0.62, 62.52, incalculable, and 11.15, respectively. The results obtained suggest that (a) analogs I and VI are potent antagonists of the pressor response of angiotensin II in normal rat, VI being the most potent antagonist thus far synthesized; (b) replacement of position 4 (Tyr) with MeTyr or position 5 and/or 8 (Ile) with Melle in [1-sarcosine,8-isoleucine]angiotensin II reduced the antagonist activity of this peptide (rabbit aortic strips and rats), indicating that steric hindrance imposed due to N-methylation in positions 4, 5, or 8 was not favorable in eliminating the initial pressor activity or prolonging the duration of action of [Sar1, Ile8]angiotensin II without reducing its antagonistic properties; (c) except II, none of the analogs showed any enhanced duration of action, suggesting that N-methylation in positions 5 or 8 did not afford protection against proteolytic enzymes; and (d) perfusion studies in cat adrenals indicated that all of these analogs are only very weak secretagogues. With the exception of [Sar1,Thr(ObetaMe)8]angiotensin II, which gave lower antagonistic properties, all other analogs had either similar antagonistic properties or were better antagonists in adrenal medulla than in smooth muscle.     

复方氨基酸乳剂的制备及稳定性研究

本实验研制了由平衡氨基酸、中碳链甘油三酯(MCT)等组成的,供口服用的复方营养乳剂。结果,除主药外,以含4% PF-SE 的乳化剂和加入适量抗氧剂、防腐剂及稳定剂(β-CD)的处方为最好。