Pepsin films

As with all delicate equipment, the pH meter and electrode must receive proper care and maintenance. All electrodes should be kept clean and stored in solutions suggested by manufacturers. Glass electrodes are fragile and expensive, so they must be handled with care. If pH measurements of protein solutions are often taken, a protein film may develop on the electrode it can be removed by soaking in 5% pepsin in 0.1 M HC1 for 2 hours and rinsing well with water. 

These surface films of proteins are not infrequently formed on solutions in which the proteins are quite soluble, so that it may be argued that the protein has been altered or denatured by its unfoldment in the surface film, in such a way as to render it less soluble or completely insoluble. Whether the unfoldment and spreading of the molecule always results in the loss of solubility is not proven Gorter has been able to remove pepsin from a surface on which it has been spread by means of a fine net pulled up through the surface, and subsequently dissolved the material in water, finding that it retained its normal properties, including proteolytic activity. 

Two experimental results indicate that there is an adsorption energy barrier related to the interfacial pressure. First, the presence of an energy barrier becomes evident only after an interfacial pressure of 0.1 mN m-1 is attained (Table II). In the second experiment, different compounds were spread at the air/water interface and the rate of adsorption of pepsin and lysozyme were measured under conditions where charge effects were minimized (MacRitchie and Alexander, 1963a). It was found that the rates of adsorption for these proteins were independent of the nature of the surface film and depended only on the surface pressure. 

Problems (and solutions) include inaccurate and/ or unstable pH readings caused by crosscontamination (rinse electrode assembly with distilled water and blot dry between measure-ments) development of a protein film on the surface of the electrode (soak in 1% w/v pepsin in 0.1 mol L-1 HCI for at least an hour) deposition of organic or inorganic contaminants on the glass bulb (use an organic solvent, such as acetone, or a solution of 0.1 mol L 1 disodium ethylenediamine-tetraacetic acid, respectively) drying out of the internal reference solutions (drain, flush and refill with fresh solution, then allow to equilibrate in 0.1 mol L 1 HCI for at least an hour) cracks or chips to the surface of the glass bulb (use a replacement electrode). 

The application of low-temperature techniques to the investigation of protein spectra in the ultraviolet region was initiated by Lavin and Northrop (1935) who investigated the ultraviolet absorption spectra of pepsin, serum albumin, and ovalbumin in glycerol, and showed that the fine structure of the protein spectrum was enhanced at — 100°C. Preliminary reports of similar work have been published by Randall and Brown (1949) on thin films of sublimed tryptophan and phenylalanine at 90°C., and by Sinsheimer et al. (1949) for tryptophan at 77.6°K. Loof-bourow and his coworkers (Sinsheimer et al., 1950) have begun publication of a series of papers reporting much more comprehensive work on the influence of low temperature on the spectra of amino acids and proteins in thin films and in solid solution. Beaven et al. (1950) have reported a few results on thin Aims of the aromatic amino acids. 

Bresler expected that polycondensation should take place between stearic aldehyde 27 at the interface and either of the amines 28 or 29 which was present in the subphase, to produce polyaminals 30. > The former combination was supposed to produce 2-D Hnear polymers (The authors a linear polymer in 2-D confinement), and actually gave rise to a product that exhibited no elastic properties and only an increased viscosity. In contrast, the latter combination afforded a product which behaved as a brittle soUd that was ascribed to a 2-D network (The authors an irregularly crosslinked monolayer in 2-D confinement). The copolymerization of pepsin with either formaHn or a diamine was also studied, and this resulted in elastic, rubber-like films. In none of these cases were any details of degree of polymerization nor any structural analysis of the products provided, however. 

Figure 3-8. Results of Norman Heatley s experiment to determine whether a layer of mucus impedes diffusion of pepsin through agar. The length of each bar represents the amount of pepsin found in each of six discs in a pile after diffusion from the top disc (1) when that disc was or was not separated from the others by a film of concentrated mucus between discs 1 and 2. (From Heatley NG. Mucosubstance as a barrier to diffusion. Gastroenterology 37 313-317, 1959.)...

Due to the inherent technical difficulties, the chemical reactions of spread protein monolayers have not been extensively investigated. Schulman and Hughes (64) report that the injection of ch3rmotrypsin under a casein film causes hydrolysis of the film as evidenced by a change in the film potential. They also report that pepsin injected under a casein film will digest the film at pH 2. 

Monolith capillary microreactor Pepsin A Entrapment Pepsin-encapsulated film was prepared by a sol-gel method on-line digestion of insulin chain beta and lysozyme 100% recovery for insulin chain (3-amino acid sequence 73% recovery for the lysozyme amino acid sequence [139]... 

Lysozyme and pepsin adsorbed on gold, aluminium and titanium have also been investigated. Similar changes occur in the infrared spectra upon adsorption of these proteins onto metals. The Amide I bands are shifted approximately 20 cm" towards higher frequencies and the Amide II bands are shifted to lower frequencies due to the decreased degree of hydrogenbonding in the protein films. 

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