Effect of pH on protein solubilty and denaturation

Effect of pH on protein solubilty and denaturation

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I'm a little bit confused about the influence of pH on proteins.
Increasing or lowering the ph will give the protein a net positive or net negative charge, respectively. However a lower pH or higher pH will cause the protein to denaturate --> more hydrophobic amino acids will become free to interact between different proteins --> less solubilty.
let's clarify my question
take a look at the following figure:

this figure indicates that a lower or higher pH then the Pi will increae the protein solubility.
However the Introduction to this article, for example, cites papers to support the following statement:

Changes in pH are shown to trigger formation of amyloid fibers and aggregation

the figure and the article are inconsistent in my opinion, so I'm definitly missing something. The first figure indicates that protein solubilty will increase when the pH becomes higher (then the pI). However the article statement indicates that an increase in pH will cause the proteins to aggregate, so lowering solubilty right?

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.


This paper treats the free energy contribution of ionizable groups to protein stability. A method is presented for the calculation of the pH dependence of the denaturation free energy of a protein, which yields results that can be compared directly to experiment. The first step in the treatment is the determination of the average charges of all the ionizable groups in both the folded and unfolded protein. An expression due to Tanford then relates the pH dependence of the unfolding free energy to the difference in net charge between the two states. In order to determine absolute rather than relative unfolding free energies, it is necessary to calculate the total contribution of ionizable groups to protein stability at some reference pH. This is accomplished through statistical mechanical treatment similar to the one used previously in the calculation of pKas. The treatment itself is rigorous but it suffers from uncertainties in the pKa calculations. Nevertheless, the overall shape of experimentally observed plots of denaturation free energy as a function of pH are reasonably well reproduced by the calculations.

A number of general conclusions that arise from the analysis are (1) knowledge of titration curves and/or effective pKa values of ionizable groups in proteins is sufficient to calculate the pH dependence of the denaturation free energy with respect to some reference pH value. However, in order to calculate the absolute contribution of ionizable groups to protein stability, it is necessary to also know the intrinsic pKa of each group. This is defined as the pKa of a group in a hypothetical state of the protein where all other groups are neutral. (2) Due to desolvation effects, ionizable groups destabilize proteins, although the effect is strongly dependent on pH. There are however, strongly stabilizing pairwise Coulombic on the surface of proteins. (3) Plots of stability versus pH should not be interpreted in terms of a group whose pKa corresponds to the titration midpoint, but rather to a group with different pKas (that correspond approximately to the titration end points) in each state. (4) Any residual structure in the GuHCl-denatured state of proteins appears to have little effect on the pH dependence of stability. (5) pH-dependent unfolding, for example to the "molten globule" state, appears due to individual groups with anomalous pKas whose locations on the protein surface may determine the nature of the unfolded state.

Types of Denaturation

Denaturation can be classified based on the agent that causes a protein to lose its secondary, tertiary or quaternary structure. Hydrogen bonds, ionic interactions, hydrophobic interactions and Van der Waal’s forces are involved in holding these structures together. Such non-covalent interactions are disrupted by a number of factors including heat, radiation, organic solvents, acids, bases and salts because they can alter hydrophobic interactions in the interior of the protein, affect hydrogen bonding and interfere with ionic interactions.

Type I: Denaturation by change in pH

The pH of a solution has an important effect on protein structure because it changes the number and nature of hydrogen bonds and ionic interactions that take place between different amino acids. At physiological pH, many amino acid side chains are charged due to the loss or gain of a hydrogen ion.

In fact, most amino acids exist as a zwitterion within cells.

Two isomers of Alanine, where the carboxylic acid group loses a hydrogen atom and obtains a negative charge and the amine group gains a proton to become positive. The net charge on the molecule remains zero. In addition to these two charges on every amino acid, the side chain can also be polar or charged.

Aspartic acid, glutamic acid, arginine, histidine and lysine are examples of amino acids that contain an extra charge at physiological conditions. However, the charge on these amino acids is dependent on pH. For instance, histidine is usually positively charged in a cell. However, it can occur in four different forms based on the pH of the solution. Due to this, it can either have a net charge of -1, 0, +1 or +2. This implies that the pH will determine the nature and number of ionic bonds a histidine residue can form with other amino acids.

When a hydrogen atom is covalently bound to a highly electronegative atom such as oxygen or nitrogen, it obtains a partial positive charge. Due to the small size of the hydrogen atom, this partial charge is sufficient to create a high charge density that can exert a strong pull on a lone pair of electrons on another atom.

This attraction forms the basis of a hydrogen bond. It is sensitive to changes in pH because a change in hydrogen ion concentration can alter the nature of functional groups. For instance, amino acids such as asparagine, cysteine or tyrosine contain polar groups in their side chains. These atoms can interact with each other through hydrogen bonds based on their spatial proximity and the orientation of different side chains.

However, as pH changes, these polar groups can get protonated or deprotonated, changing their ability to form hydrogen bonds. In addition, the formation of an alpha helix or a beta-pleated sheet involves hydrogen bond formation between the carboxylate and amine groups of every amino acid. If a large number of such interactions are interrupted, the protein unfolds and becomes denatured.

Finally, pH affects the overall charge of a polypeptide and this has an impact on protein solubility. If the net charge on a protein becomes zero, it can aggregate and precipitate.

Type II: Chemical Denaturation

Certain chemicals and organic solvents can cause protein denaturation. Organic solvents disrupt protein structure because most proteins sequester their hydrophobic residues towards the center of the molecule when they fold into their unique shape.

Essentially, the 3-D structure of a polypeptide optimizes the energy of the molecule by lowering the interaction of hydrophobic side chains with an aqueous medium. The presence of chemicals like benzene or ethanol changes these interactions and can occasionally lead to the protein ‘flipping’ where the internal residues are presented on the outside with the loss of structure and function.

Detergents and other amphipathic molecules can also be particularly damaging. These molecules have the same effect by disrupting hydrophobic interactions and hydrogen bonds. The frothing seen during protein extraction in laboratories as well as the foam associated with household detergents are, in part, due to their denaturing effect on proteins. Heavy metals and high salt concentrations can affect the formation of ionic bonds, while chaotropic agents like urea can have an extensive disruptive effect on the hydrogen bond network.

Type III: Denaturation by Heat and Radiation

When the temperature of a biological sample is increased, it leads to an overall increase in the kinetic energy of every atom and an increase in the entropy of the system. All the atoms and molecules start to have higher energy collisions with one another as well as increased translational, rotational and vibrational motion.

This reduces the strength of hydrogen bonds and weakens the influence of non-polar hydrophobic interactions. This is one of the reasons why a high fever is used by the body to fight infections. It is an attempt to retard or arrest the growth of microorganisms so that the immune system can clear the pathogens.

However, a sustained high fever can be detrimental to the proteins within the host as well which is why temperatures beyond 104° Fahrenheit (40° Celsius) are considered dangerous. Prolonged exposure to radiation from the sun also leads to loss of protein structure. Sunburns and cataracts in the eye are just two examples of its deleterious effects. Radiation from other sources (such as an X-ray machine or from radioactive materials) can also cause damage to proteins and result in a number of ailments.

Physical Properties of Proteins

  1. Colour and Taste
    Proteins are colourless and usually tasteless. These are homogeneous and crystalline.
  2. Shape and Size
    The proteins range in shape from simple crystalloid spherical structures to long fibrillar structures. Two distinct patterns of shape
    have been recognized :
    A. Globular proteins- These are spherical in shape and occur mainly in plants, esp., in seeds and in leaf cells. These are bundles formed by folding and crumpling of protein chains. e.g., pepsin, edestin, insulin, ribonuclease etc.
    B. Fibrillar proteins- These are thread-like or ellipsoidal in shape and occur generally in animal muscles. Most of the studies regarding protein structure have been conducted using these proteins. e.g., fibrinogen, myosin etc.
  3. Molecular Weight
    The proteins generally have large molecular weights ranging between 5 × 103 and 1 × 106. It might be noted that the values of molecular weights of many proteins lie close to or multiples of 35,000 and 70,000.
  4. Colloidal Nature
    Because of their giant size, the proteins exhibit many colloidal properties, such as Their diffusion rates are extremely slow and they may produce considerable light-scattering in solution, thus resulting in visible turbidity (Tyndall effect).
  5. Denaturation
    Denaturation refers to the changes in the properties of a protein. In other words, it is the loss of biologic activity. In many instances the process of denaturation is followed by coagulation— a process where denatured protein molecules tend to form large aggregates and to precipitate from solution.
  6. Amphoteric Nature
    Like amino acids, the proteins are amphoteric, i.e., they act as acids and alkalies both. These migrate in an electric field and the direction of migration depends upon the net charge possessed by the molecule. The net charge is influenced by the pH value. Each protein has a fixed value of isoelectric point (pl) at which it will move in an electric field.
  7. Ion Binding Capacity
    The proteins can form salts with both cations and anions based on their net charge.
  8. Solubility
    The solubility of proteins is influenced by pH. Solubility is lowest at isoelectric point and increases with increasing acidity or alkalinity. This is because when the protein molecules exist as either cations or anions, repulsive forces between ions are high, since all the molecules possess excess charges of the same sign. Thus, they will be more soluble than in the isoelectric state.
  9. Optical Activity
    All protein solutions rotate the plane of polarized light to the left, i.e., these are levoratotory.

Chemical Properties of Proteins

  1. Hydrolysis
    Proteins are hydrolyzed by a variety of hydrolytic agents.
    A. By acidic agents: Proteins, upon hydrolysis with conc. HCl (6–12N) at 100–110°C for 6 to 20 hrs, yield amino acids in the form of their hydrochlorides.
    B. By alkaline agents: Proteins may also be hydrolyzed with 2N NaOH.
  2. Reactions involving COOH Group
    A. Reaction with alkalies (Salt formation)
    B. Reaction with alcohols (Esterification)
    C. Reaction with amines
  3. Reactions involving NH2 Group
    A. Reaction with mineral acids (Salt formation): When either free amino acids or proteins are treated with mineral acids like HCl, the acid salts are formed.
    B. Reaction with formaldehyde: With formaldehyde, the hydroxy-methyl derivatives are formed.
    C. Reaction with benzaldehyde: Schiff ‘s bases are formed
    D. Reaction with nitrous acid (Van Slyke reaction): The amino acids react with HNO2 to liberate N2 gas and to produce the corresponding α-hydroxy acids.
    E. Reaction with acylating agents (Acylation)
    F. Reaction with FDNB or Sanger’s reagent
    G. Reaction with dansyl chloride
  4. Reactions involving both COOH AND NH2 Group
    A. Reaction with triketohydrindene hydrate (Ninhydrin reaction)
    B. Reaction with phenyl isocyanate: With phenyl isocyanate, hydantoic acid is formed which in turn can be converted to hydantoin.
    C. Reaction with phenyl isothiocyanate or Edman reagent
    D. Reaction with phosgene: With phosgene, N-carboxyanhydride is formed
    E. Reaction with carbon disulfide: With carbon disulfide, 2-thio-5-thiozolidone is produced
  5. Reactions involving R Group or Side Chain
    A. Biuret test
    B. Xanthoproteic test
    C. Millon’s test
    D. Folin’s test
    E. Sakaguchi test
    F. Pauly test
    G. Ehrlich test
  6. Reactions involving SH Group
    A. Nitroprusside test: Red colour develops with sodium nitroprusside in dilute NH4.OH. The test is specific for cysteine.
    B. Sullivan test: Cysteine develops red colour in the presence of sodium 1, 2-naphthoquinone- 4-sulfonate and sodium hydrosulfite.

Mini Review Important Factors Influencing Protein Crystallization

The solution of crystallization problem was introduced around twenty years ago, with the introduction of crystallization screening methods. Here reported some of the factors which affect protein crystallization, solubility, Concentration of precipitant, concentration of macromolecule, ionic strength, pH, temperature, and organism source of macromolecules, reducing or oxidizing environment, additives, ligands, presence of substrates, inhibitors, coenzymes, metal ions and rate of equilibration. The aim of this paper to give very helpful advice for crystallization.


X-ray crystallography has provided 3D structures of thousands of proteins. In spite of these advances, many factors continue to be problem that can lead to unsuccessful proteins crystallization. We always know theoretical pI, molecular weight and amino-acid composition, while pH and salt concentration are some of the variables that can be expected from other similar known structure. Yet, a protein behavior depends very much on the environment it is in.

Proteins are generally present in a biological sample as their native state. They are very often associated with other proteins and integrated into large complexes. In most cases they are not soluble in their innate state after isolation as result of that must be denatured to help solubilization and Crystallization. Initial protein crystal commonly need to screen conditions using little as possible to improve the optimization methods for protein crystallization.

Crystallization is thermodynamically process. Once begin, will continue under kinetic control until super saturation is lost. The crystallization process affected by physical conditions of the solution, solution solubility, the presence of impurities, nucleation, solution saturation and degree of super saturation, crystal growth, including solution composition, pH and temperature, and to date is not fully understood.

Prediction of secondary structure, solubility, domain organization, stability, signal peptide, hydrophobicity and pH, can provide useful information for crystallization strategies and salt prediction tools it need to be develop. in general proteins has low molecular weight easier for crystallization than high molecular weight, single-domain easier for crystallization than multi-domain and an oligomeric state multimer more likely to crystallize than a monomer high molecular weight.

Buffer is most straight forward way to make a crystallization problem by effect the protein behave. There are many rules and different protein crystallization methods, some of it has been developed during the recent years. However, Protein crystallization still represents a great challenge for bio crystallography. The cumulation of crystallization information in crystallization databases and in structural articles it allow us to design crystallization experiments depending on the character of the protein. Because there is no ideal procedure for crystallization, our article discusses the crystallization problem try to simplify the composition of the crystallization and give some solution advice.

Protein solubility

Protein solubility is a common complex interaction problem between the physiochemical nature of the proteins, rate of protein synthesis, amino acid composition, the Protein concentration, type of salt present in the buffer, the concentration of the salt used, ionic strength, pH, temperature, osmotic pressure, cellular location of expression, and cellular tools or chaperones are all important in protein solubility. Protein solubility may decrease in the presence of complexes with lipids, nucleic acids or other nonprotein.

It is important to select better option for tag in the target proteins. Unfortunately, there is no consolidated system for rapid screening to differentiate the best tag.

Addition L-Arg and L-Glu at 50 mM to the buffer can increase solubility up to 8.7 times, because they can in preventing protein aggregation and precipitation as well as increase the long-term stability also it did not prevent specific protein-protein and protein-RNA interactions [1].

Normally when there is solubility issues, the first thing we need to check it the buffer composition, varying pH and salt strength it can make a big difference to solubility. Then try solubilising agents, e.g NDSBs or detergents. Protein solubility can improved by selecting only a soluble domain for expression, and delete the hydrophobic domain. Moreover there are solubility prediction tools in the internet it can be useful.

In order to be sure target protein is soluble culture need to be lysate (lysozime 1mg/ml on ice 1hour, it should be under non denaturation condition) and centrifugation at 35000 x g. then use an aliquot of supernatant for SDS-PAGE. In case of protein is soluble well found in the supernatant.


Detergents are commonly used at concentrations of 1–4%. It useful in solubilizing by disrupt hydrophobic interactions between and within proteins. The detergent used for solubilization does not requisite to be the same for purification and crystallization, also the presence of various detergents perhaps inhibit the activity of different proteases. When despite all efforts not work then it needs denaturation and solubilization of the target protein. This is done by a denaturing agent e.g. guanidine or urea under reducing conditions (

20 mM DTT). A western blot necessary for the supernatant before and after the high-speed centrifugation will give an indication of how much detergent was effect in the supernatant (membrane).

Reducing agents

Reducing agents are generally used in sample preparation to cleave disulfide bond DTT, DTE and β-ME are more commonly reducing agents used. The aim of using these agents to protect proteins from precipitating and aggregating due to oxidative crosslinking and to reduce protein aggregation that may inhibit crystallization, Reducing agents can interact with metals within protein sample and that is not good for crystallization in this case. BME is particularly sensitive to cobalt, copper and many phosphate buffers while DTT is sensitive to nickel. But until now no specific article show different between the DTT, DTE and β-ME as well as in which crystallization step is better to be use.

Isoelectric point (pI), pH & Temperature

The pH influence on protein solubility, different proteins are soluble at different pH values, at high pH protein soluble (deprotonate), low pH protein soluble (Protonated) and at isoelectric point: protein aggregates. Acidic proteins has pI lower than 7, more likely to crystallize around one pH unit above their pI, while basic proteins more likely to crystallize lower than their pI about 1.5–3 pH units [2]. Generally, protein aggregation or precipitation when the solubility decrease at pH close to the isoelectric point (pI). It better to move pH away from the pI to increase protein solubility and Super saturation. At low salt concentration the electrostatic repulsions well decrease when pH is different from pI, this well likely induce PEG to effect depletion attraction. The depletion attraction due to polymer is more effective when the protein net charge is lower.

To screen favor attraction between macromolecules and protein charges it better to increasing ionic strength or gently moving pH closer to pI. The most critical things is pI we need to make Sure the protein having net charge close to zero and it soluble, and this depend on some paper advice.

Actually, temperature is important parameter for crystallization, it is better be screened. Temperature influences crystal growth and nucleation by changing super saturation as well as solubility of the sample. For instance, pH of Tris buffer is susceptible to temperature changes. However, temperature can useful at low ionic strengths by effects the amplified. Proteins very often show several crystal polymorphs as result of different temperature. Lowering the growth inducing temperature, this lessening the rate of protein synthesis and usually more soluble protein is obtained. Because the temperature can affects solubility is better to cool the Crystal solutions to prevent protein degradation.

Optimum salt concentration

Different NaCl concentrations with different pH levels it was use for crystallization. The effective of salt concentration in a crystallization solution can be prognosticate before performing in the experiment, because it is depended on the buffer pH and the pI of the protein. It is important for successful crystallization to keep the salt concentrations neither too low nor too high in the protein solution, highly salt concentration may stabilize the protein buffer or decrease the protein solubility lead to precipitation. Then crystallization happens in the drop in which the components are concentrated through water loss. If the original proteins as well as reservoir solution do not contain enough salt, the concentration in the drop does not reach the marginal concentration level.

Commonly, the increase of precipitant concentration in crystal solutions is an effective technique adopted to lowering the solubility of proteins. The salt it can stabilizes the water structure (Kosomtropic) or disrupts the water structure (Chaotropic). In addition, the solubility of the proteins drop at high concentrations of salt as well as solubility of the proteins increases at low concentrations of salt. The optimal salt concentration will slowly dehydrate our drop as well as increase the concentration of our protein slowly.

The marginal ionic strength become high when the difference between the pI of the proteins and the pH of the buffer is large, which is agreement with the idea of the electrostatic screen effect of a salt [3].

Multi subunit proteins or virus it is large macromolecules, highly salt concentrations are not effective to promote enough interactions to induce crystallization while the Polyethylene glycol (PEG) is a commonly used as precipitant in protein crystallization is indispensable to induce crystallization.

Crystallization of iron protein under anaerobic condition may facing precipitation problem, if the oxidation of the protein causing the precipitation try to add sodium dithionite to preserve the reduced state, also is better to change the ratio in the crystallization drop by higher the amount of the precipitant.

Expression system

E coli is first choice for structural biologists to produce protein for X-ray crystallography, but sometime unsuitable to express membrane proteins in bacterial expression systems because the system cannot provide the post-translational modifications, the folding machinery and specific lipid environment. For eukaryotic membrane protein better to express in Saccharomyces cerevisiae, Pichia pastoris, Sf9 insect cells [4] and human embryonic kidney.

Improve the expression level

In case of poor expression it need to be sure the protein is not toxic for the cell. Choosing a smaller fragment of the target protein can improve the expression levels and solubility, because E. coli may not express well very large proteins > 70 kDa. Using strains carrying mutations which remove the production of proteases can occasionally enhance accumulation by diminution proteolytic degradation, also reducing the growth temperature will produce protein slow but at the same time slower proteolytic degradation. Using special media containing trace metals (high density culture media) it can increase protein yield 10 time over LB.

Co-expression of foldases proteins e.g. peptidyl prolyl cis/trans isomerases (PPI’s), disulfide oxidoreductase (DsbA), disulfide isomerase (DsbC) and protein disulfide isomerase (PDI) with the target protein may lead to highly levels of protein solubility. The other advantages for this enzymes assist the formation of disulfide bonds which does not happen in the reducing environment an also reduced proteolysis

Commonly challenging and important task, protein aggregation and precipitation is often happen when increasing a protein concentration in the buffer to the required level.

Size exclusion chromatography

Is useful for further purification but some time is better to use new column for purification. The shape of the chromatogram and retention time provides.

High homogeneity and purity of the sample are crucial for the crystallization to be successful, the presence of different aggregates or oligomeric forms in the protein solution it well effect, we need to use Dynamic light scattering (DLS) to check that or small-angle X-ray scattering (SAXS).

Folding and stability

‘Natively unfolded’ proteins are some proteins unfolded in any physiological conditions, and this kind may better to co crystallize with other protein. UN folding, while highly beta strand are more likely to form amyloid-like aggregates, but both of them can be crystallize PDB (1aos--1by3). There is no direct correlation between percentage of helix secondary structure as well as beta strand and stability. However, proteins with high alpha-helical protein habitually are less problematic upon. There are more hydrophobic contacts in Beta sheets, misfolding or mutation might result in exposure of hydrophobic residues. In this case, proteins aggregate to inhibit hydrophobic exposure as well as avoid entropic penalty. Beta sheets are stabilized through hydrogen bonding as well as hydrophobic contacts, while Alpha helices are predominantly stabilized through hydrogen bonding. Thus sheet is a little inferior in terms of stability.

Addition of co-factors or prostethic groups (such as a vitamin, lipid, or inorganic like a metal ion) which are essential for protein stability or for proper folding, but must be not fluctuation the pH in the medium during growth, this leads to the accumulation of the target protein osmoprotectants in the cell, which stabilize the protein structure.

Biophysical methods like CD spectroscopy and other differential scanning fluorometry (DSF) can be used for characterizing the stability of the protein in different buffers, pH and in the presence of different ligands to make sure that the protein is correctly folded. There is several fold recognition websites (FOLDpro, RF-Fold, SSHMM, THREADER, BLASTLINK, SSEARCH, PSI-BLAST and HMMER) used to predict the protein fold.


When the native protein fails to crystallize against

300500 crystallization conditions it was propose genetically modified proteins whether by truncations or point mutations might better for crystallization.

Mutants of Tyr →Phe and Thr→Val more stable than the wild-type protein, but have little effect on the conformation of the protein [5]. However, it has been reported that point mutations spectacular affect the amount of aggregate formation in number of protein systems these include the interferon-γ, P22 tailspike protein, colicin A, single-chain antibodies, immunoglobulin domains, and interleukin-1β [6]. However, it is still not clear what mutations are likely to be helpful in crystallization (Figure 1).

Figure 1:

superdex 75 A. New superdex 75 B. Old superdex 75 as we can see new superdex 75 separate the protein to three pick, one is aggregation protein, two is dimer protein, three is monomer, while the old superdex 75 cannot separate them and in this case is difficult to be crystal because the crystal condition for dimer is not same to the monomer condition rather then that aggregation it can be crystal.

Cysteine and histidine predominantly react with reagents prepared for the other and for other amino acid side chains as well, that why it is problematic during crystallization. Some domain and cloning are affecting the solubility this checking before expend time on buffer optimization.


Sometime one protein can create different crystal, but that not mean all of them they are stable, it can just be kinetically favored. There is two kind of nucleation: heterogeneous is the most common, occurs by interface of different composition and homogeneous as high purity crystal. The nucleation rates it can be increased by increasing the super saturation, solubility, viscosity and decreases temperature and solid–liquid interfacial tension.


In ethanol, the assist of peptide group burial to protein stability would be increase and the contribution of non-polar group burial would be decrease. Because α-helices are expected to be stable structures in ethanol, ethanol used to increase α-helix formation in proteins and peptides [7], also ethanol and methanol can lowers the thermal denaturation temperature.


Often use in crystallization to protect the proteins as cryosolvent because antifreeze properties and to enhance their solubility by stabilizing the conformation, sugar protein specially affected by glycerol because of ribonuleotides bonds. But some time glycerol work as an antinucleation agent in crystallization.

TRIS & HEPES buffers

The differences in the influence of the buffers could be attributed neither to disparity in the amounts of de-protonated buffer ions nor to disparity in ionic strength. Tris and HEPES being positively charged at pH 7.0. Protein activity increased in different buffer by following order: citrate MES > HEPES > TRIS > phosphate [8]. Tris as well as HEPES prevent the auto-oxidation. HEPES accelerated the degeneration rate of the oxidant and peroxynitrite. The exist of as much as 100 mM NaCl enhance the reversibility and stability of unfolding transitions in Hepes buffer. The crystal structure obtain from HEPES buffer was more similar to the active conformation.

Proteins in L-arginine and Tris buffer at pH 7.4 stay stable against aggregation for longer periods of time. Tris is Good’s buffers bind to the DNA not only by electro static interactions but also by hydrogen bonds primarily to the pyrimidine or purine rings [9]. Tris decrease the metal ion–catalyzed oxidation of Cys, consequently, Cys being available for interaction with the other reagent.


In this review presents important factors that affect protein crystallization and ways to improve the results, but more needs to be done to better understand. Some proteins have ability to make crystal within initial concentration less than 1 mg per ml. Right working sequence and buffer well make all the difference.


This work was supported by Ministry of Science and Technology of China and the CAS-TWAS President’s PhD Fellowship. All authors declare no conflict of interests.


Comparing protein solubility in PEG-8000 and ammonium sulfate

To compare the solubility measured using PEG-8000 with that measured using ammonium sulfate, we evaluated the log S0-values from the two fits. Fig.ਅ shows the plot of log S0 ∗ obtained with ammonium sulfate (log S0 ∗ (NH4)2SO4) versus log S0 obtained with PEG-8000 (log S0 PEG). A remarkably strong correlation between the solubility results for ammonium sulfate and those for PEG-8000 is seen. This suggests that log S0 ∗ (NH4)2SO4 is a parameter that is related to protein solubility in the absence of precipitant. Because log S0 PEG can be used to estimate solubility in the absence of buffer, the correlation of log S0 PEG with log S0 ∗ (NH4)2SO4 suggests that log S0 (NH4)2SO4 can be used qualitatively to determine differences in solubility.

Comparison of the solubility data obtained in ammonium sulfate and PEG-8000. Log S0-values obtained from PEG-8000 precipitations are plotted against Log S0 ∗ -values from ammonium sulfate precipitations for all of the proteins. The data correlate strongly, suggestingਊ relationship between solubility results obtained with PEG-8000 and ammonium sulfate. α-Lactalbumin is shown as an open diamond and is excluded from the fit.

The solubility of α-lactalbumin warrants further discussion. In the case of PEG precipitation, α-lactalbumin is predicted to have the highest solubility of the proteins used in this study. This is not surprising given that α-lactalbumin is present in high concentrations in bovine milk (49) and the fact that we are able to make stock concentrations of α-lactalbumin that are in excess of 100 mg/mL. α-Lactalbumin has a β-value in PEG-8000 that is intermediate of the slopes observed for the other proteins. In the case of ammonium sulfate, α-lactalbumin is predicted to have the lowest solubility among the proteins studied, and the slope observed with α-lactalbumin is a clear outlier. It is the smallest slope observed: 13-fold lower than the average slope, and fivefold lower than the next-closest slope in ammonium sulfate. This suggests that ammonium sulfate is not as effective as a precipitant for α-lactalbumin as it is for the other proteins. This may be due in part to the high surface charge on α-lactalbumin: two thirds of the exposed surface residues carry a charge at pH 7 (data not shown) and nearly a third of the accessible surface area is charged (see Table 3 , column 9). We previously suggested that ammonium sulfate may underestimate the contribution of charged surface residues to protein solubility (16). This likely is related to the mechanism by which ammonium sulfate lowers protein solubility (i.e., increasing surface tension and competing for waters with the protein surface). The high level of charged surface area on α-lactalbumin (roughly equal amounts positive and negative) likely affects the ability of ammonium sulfate to act as a precipitant. The kosmotropic carboxylates on the protein surface compete strongly for water molecules with the sulfate ions, and the chaotropic amino and guanidino groups may lower the water surface tension at the protein water interface, partially opposing the effect of ammonium sulfate. Due to the unique nature of the salting-out curve of α-lactalbumin, the ammonium sulfate data were fit without α-lactalbumin in subsequent correlations.

Table 3

Protein properties and surface properties used for correlations

ProteinMolecular mass (kDa)Amino acidspI ∗ Charge ∗ Absolute charge ∗ Fraction of ASA †
Human serum albumin66.55856�.212.20.590.410.270.120.14

In an attempt to determine the intrinsic factors that influence protein solubility, we looked at several intrinsic protein properties and examined them with respect to protein solubility by comparing them with log S0-values obtained in this study. We looked at fundamental properties of the protein, such as size (molecular mass) and net charge. We also looked at properties of the surface of the protein, including polarity and charge, by determining the fraction of the surface area of the protein that was polar, nonpolar, charged, negatively charged, or positively charged. By looking at the correlation of these properties with protein solubility, we were able to determine their relative importance for determining protein solubility.

Correlation of molecular mass and net charge with solubility measurements

To investigate the contribution of intrinsic factors to protein solubility (see Table 3 , columns 1𠄶), we plotted log S0-values for PEG-8000 and ammonium sulfate versus molecular mass ( Fig.ਆ , A and B), net charge ( Fig.ਆ , C and D), and the absolute value of the net charge ( Fig.ਆ , E and F). Linear fits were made to the data, and R 2 values are given. Because the mechanism of PEG precipitation is related to the excluded volume, solubility may increase with protein size or molecular mass however, no correlation with molecular mass was observed. In general, the solubility of a given protein is at a minimum near the isoelectric point (pI) and increases with the absolute value of the net charge (13,50). To assess whether net charge plays a role in determining the solubility of a group of proteins, we plotted the net charge and absolute value of net charge versus log S0. A weak correlation was observed in all four cases. In the case of the absolute value of net charge, a weak positive correlation was observed, suggesting that with an increasing positive or negative net charge, protein solubility increases. For net charge versus pH, a weak negative correlation was observed. This suggests that, on average for this set of proteins, negatively charged proteins are more soluble than positively charged proteins however, more data points are required to determine whether this is true for a larger set of proteins.

Correlation of molecular mass and net charge with PEG-8000 and ammonium sulfate solubility measurements. Log S0-values versus molecular mass (A and B), net charge (C and D), and absolute net charge (E and F) are shown. The lines and R 2 values are from linear least-squares fits. α-Lactalbumin is shown as open diamonds and is excluded from the ammonium sulfate fits.

Correlation of the intrinsic properties of the accessible surface area with protein solubility

Because protein solubility is influenced largely by interactions between water and the protein surface, we investigated the correlation between solubility and the intrinsic properties of the surface of the proteins. The accessible surface areas (ASAs) of all atoms in the proteins were determined and the fractions that were polar, nonpolar, charged, positively charged, and negatively charged were calculated (see Table 3 , columns 7�). Fig.ਇ depicts protein solubility as a function of fraction ASA that is polar or nonpolar for PEG-8000 ( Fig.ਇ , A and B) and ammonium sulfate ( Fig.ਇ , C and D). For PEG-8000, the correlation of the percentage of polar and nonpolar surface residues with protein solubility is very poor, although the correlation is positive for polar residues and negative for nonpolar residues, as might be predicted. This suggests that the surface polarity makes a minimal contribution to protein solubility in PEG-8000. For ammonium sulfate, a better correlation was observed, but the correlation with polar and nonpolar surface residues is negative and positive, respectively, which is the opposite of what we would have expected and what was observed in PEG-8000.

Correlation of fractions of polar and nonpolar ASAs with PEG-8000 and ammonium sulfate solubility measurements. The ASA for all atoms was calculated using PDB files and either pfis (31) or NACCESS (32). Carbon and sulfur atoms are considered nonpolar, and nitrogen and oxygen atoms are considered polar. Log S0-values versus fraction polar ASA (A and C) and fraction nonpolar ASA (B and D) are shown. The lines and R 2 values are from linear least-squares fits. α-Lactalbumin is shown as open diamonds and is excluded from the ammonium sulfate fits.

The contribution of the ASA that is charged, positively charged, and negatively charged was evaluated (see Table 3 , columns 9�). Fig.ਈ depicts the correlations of PEG-8000 and ammonium sulfate with the fraction of charged ( Fig.ਈ , A and B), positively charged ( Fig.ਈ , C and D), and negatively charged ( Fig.ਈ , E and F) ASA. We find a strong correlation between solubility in PEG-8000 and fraction of charged ASA and a more moderate correlation for ammonium sulfate. A correlation between solubility and the fraction of positively charged ASA was not observed for either PEG-8000 or ammonium sulfate however, a very strong correlation was observed between solubility and the fraction of negatively charged ASA in both PEG-8000 and ammonium sulfate. This strong correlation suggests that negatively charged surface area plays a significant role in determining protein solubility. This is supported by our previous findings that aspartic and glutamic acids contribute more favorably to protein solubility than do any of the other 18 amino acids (16). To understand the difference in contribution to protein solubility of negative versus positive charges, one needs to understand the properties of negatively and positively charged groups in proteins. Negatively charged groups in proteins include the kosmotropic carboxylate groups of aspartic acid and glutamic acid residues. Positively charged groups include the chaotropic amino and guanidino groups of lysine and arginine. In studies on ions in solution, Collins (26,51�) described a Hofmeister series dependence for hydration of ions in solution. Collins showed that kosmotropes are highly hydrated and bind water more tightly than water binds itself, whereas chaotropes bind water more weakly than water binds itself and remain largely unhydrated in solution. Therefore, the differential contribution to the solubility of negative and positive groups on the protein surface appears to be due to the differential hydration of the carboxylates that bind water tightly and the amino and guanidino groups that bind water weakly.

Correlation of the fraction of ASA that is charged (A and B), positively charged (C and D), and negatively charged (E and F) at pH 7.0 with PEG-8000 and ammonium sulfate solubility measurements. The ASA for all atoms was calculated using PDB files and either pfis (31) or NACCESS (32). The oxygen atoms glutamic acid and aspartic acid side chains and the C-terminus are considered negatively charged, and the nitrogen atoms from arginine and lysine side chains and the N-terminus are considered positively charged at pH 7. The lines and R 2 values are from linear least-squares fits. α-Lactalbumin is shown as open diamonds and is excluded from the ammonium sulfate fits.

3.4 Proteins

Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective they may serve in transport, storage, or membranes or they may be toxins or enzymes. Each cell in a living system may contain thousands of proteins, each with a unique function. Their structures, like their functions, vary greatly. They are all, however, polymers of amino acids, arranged in a linear sequence.

Types and Functions of Proteins

Enzymes , which are produced by living cells, are catalysts in biochemical reactions (like digestion) and are usually complex or conjugated proteins. Each enzyme is specific for the substrate (a reactant that binds to an enzyme) it acts on. The enzyme may help in breakdown, rearrangement, or synthesis reactions. Enzymes that break down their substrates are called catabolic enzymes, enzymes that build more complex molecules from their substrates are called anabolic enzymes, and enzymes that affect the rate of reaction are called catalytic enzymes. It should be noted that all enzymes increase the rate of reaction and, therefore, are considered to be organic catalysts. An example of an enzyme is salivary amylase, which hydrolyzes its substrate amylose, a component of starch.

Hormones are chemical-signaling molecules, usually small proteins or steroids, secreted by endocrine cells that act to control or regulate specific physiological processes, including growth, development, metabolism, and reproduction. For example, insulin is a protein hormone that helps to regulate the blood glucose level. The primary types and functions of proteins are listed in Table 3.1.

Protein Types and Functions
Digestive EnzymesAmylase, lipase, pepsin, trypsinHelp in digestion of food by catabolizing nutrients into monomeric units
TransportHemoglobin, albuminCarry substances in the blood or lymph throughout the body
StructuralActin, tubulin, keratinConstruct different structures, like the cytoskeleton
HormonesInsulin, thyroxineCoordinate the activity of different body systems
DefenseImmunoglobulinsProtect the body from foreign pathogens
ContractileActin, myosinEffect muscle contraction
StorageLegume storage proteins, egg white (albumin)Provide nourishment in early development of the embryo and the seedling

Proteins have different shapes and molecular weights some proteins are globular in shape whereas others are fibrous in nature. For example, hemoglobin is a globular protein, but collagen, found in our skin, is a fibrous protein. Protein shape is critical to its function, and this shape is maintained by many different types of chemical bonds. Changes in temperature, pH, and exposure to chemicals may lead to permanent changes in the shape of the protein, leading to loss of function, known as denaturation . All proteins are made up of different arrangements of the same 20 types of amino acids.

Amino Acids

Amino acids are the monomers that make up proteins. Each amino acid has the same fundamental structure, which consists of a central carbon atom, also known as the alpha (α) carbon, bonded to an amino group (NH2), a carboxyl group (COOH), and to a hydrogen atom. Every amino acid also has another atom or group of atoms bonded to the central atom known as the R group (Figure 3.22).

The name "amino acid" is derived from the fact that they contain both amino group and carboxyl-acid-group in their basic structure. As mentioned, there are 20 amino acids present in proteins. Nine of these are considered essential amino acids in humans because the human body cannot produce them and they are obtained from the diet. For each amino acid, the R group (or side chain) is different (Figure 3.23).

Art Connection

Which categories of amino acid would you expect to find on the surface of a soluble protein, and which would you expect to find in the interior? What distribution of amino acids would you expect to find in a protein embedded in a lipid bilayer?

The chemical nature of the side chain determines the nature of the amino acid (that is, whether it is acidic, basic, polar, or nonpolar). For example, the amino acid glycine has a hydrogen atom as the R group. Amino acids such as valine, methionine, and alanine are nonpolar or hydrophobic in nature, while amino acids such as serine, threonine, and cysteine are polar and have hydrophilic side chains. The side chains of lysine and arginine are positively charged, and therefore these amino acids are also known as basic amino acids. Proline has an R group that is linked to the amino group, forming a ring-like structure. Proline is an exception to the standard structure of an animo acid since its amino group is not separate from the side chain (Figure 3.23).

Amino acids are represented by a single upper case letter or a three-letter abbreviation. For example, valine is known by the letter V or the three-letter symbol val. Just as some fatty acids are essential to a diet, some amino acids are necessary as well. They are known as essential amino acids, and in humans they include isoleucine, leucine, and cysteine. Essential amino acids refer to those necessary for construction of proteins in the body, although not produced by the body which amino acids are essential varies from organism to organism.

The sequence and the number of amino acids ultimately determine the protein's shape, size, and function. Each amino acid is attached to another amino acid by a covalent bond, known as a peptide bond , which is formed by a dehydration reaction. The carboxyl group of one amino acid and the amino group of the incoming amino acid combine, releasing a molecule of water. The resulting bond is the peptide bond (Figure 3.24).

The products formed by such linkages are called peptides. As more amino acids join to this growing chain, the resulting chain is known as a polypeptide. Each polypeptide has a free amino group at one end. This end is called the N terminal, or the amino terminal, and the other end has a free carboxyl group, also known as the C or carboxyl terminal. While the terms polypeptide and protein are sometimes used interchangeably, a polypeptide is technically a polymer of amino acids, whereas the term protein is used for a polypeptide or polypeptides that have combined together, often have bound non-peptide prosthetic groups, have a distinct shape, and have a unique function. After protein synthesis (translation), most proteins are modified. These are known as post-translational modifications. They may undergo cleavage, phosphorylation, or may require the addition of other chemical groups. Only after these modifications is the protein completely functional.

Link to Learning

Click through the steps of protein synthesis in this interactive tutorial.

Evolution Connection

The Evolutionary Significance of Cytochrome c

Cytochrome c is an important component of the electron transport chain, a part of cellular respiration, and it is normally found in the cellular organelle, the mitochondrion. This protein has a heme prosthetic group, and the central ion of the heme gets alternately reduced and oxidized during electron transfer. Because this essential protein’s role in producing cellular energy is crucial, it has changed very little over millions of years. Protein sequencing has shown that there is a considerable amount of cytochrome c amino acid sequence homology among different species in other words, evolutionary kinship can be assessed by measuring the similarities or differences among various species’ DNA or protein sequences.

Scientists have determined that human cytochrome c contains 104 amino acids. For each cytochrome c molecule from different organisms that has been sequenced to date, 37 of these amino acids appear in the same position in all samples of cytochrome c. This indicates that there may have been a common ancestor. On comparing the human and chimpanzee protein sequences, no sequence difference was found. When human and rhesus monkey sequences were compared, the single difference found was in one amino acid. In another comparison, human to yeast sequencing shows a difference in the 44th position.

Protein Structure

As discussed earlier, the shape of a protein is critical to its function. For example, an enzyme can bind to a specific substrate at a site known as the active site. If this active site is altered because of local changes or changes in overall protein structure, the enzyme may be unable to bind to the substrate. To understand how the protein gets its final shape or conformation, we need to understand the four levels of protein structure: primary, secondary, tertiary, and quaternary.

Primary Structure

The unique sequence of amino acids in a polypeptide chain is its primary structure . For example, the pancreatic hormone insulin has two polypeptide chains, A and B, and they are linked together by disulfide bonds. The N terminal amino acid of the A chain is glycine, whereas the C terminal amino acid is asparagine (Figure 3.25). The sequences of amino acids in the A and B chains are unique to insulin.

The unique sequence for every protein is ultimately determined by the gene encoding the protein. A change in nucleotide sequence of the gene’s coding region may lead to a different amino acid being added to the growing polypeptide chain, causing a change in protein structure and function. In sickle cell anemia, the hemoglobin β chain (a small portion of which is shown in Figure 3.26) has a single amino acid substitution, causing a change in protein structure and function. Specifically, the amino acid glutamic acid is substituted by valine in the β chain. What is most remarkable to consider is that a hemoglobin molecule is made up of two alpha chains and two beta chains that each consist of about 150 amino acids. The molecule, therefore, has about 600 amino acids. The structural difference between a normal hemoglobin molecule and a sickle cell molecule—which dramatically decreases life expectancy—is a single amino acid of the 600. What is even more remarkable is that those 600 amino acids are encoded by three nucleotides each, and the mutation is caused by a single base change (point mutation), 1 in 1800 bases.

Because of this change of one amino acid in the chain, hemoglobin molecules form long fibers that distort the biconcave, or disc-shaped, red blood cells and assume a crescent or “sickle” shape, which clogs arteries (Figure 3.27). This can lead to myriad serious health problems such as breathlessness, dizziness, headaches, and abdominal pain for those affected by this disease.

Secondary Structure

The local folding of the polypeptide in some regions gives rise to the secondary structure of the protein. The most common are the α-helix and β-pleated sheet structures (Figure 3.28). Both structures are the α-helix structure—the helix held in shape by hydrogen bonds. The hydrogen bonds form between the oxygen atom in the carbonyl group in one amino acid and another amino acid that is four amino acids farther along the chain.

Every helical turn in an alpha helix has 3.6 amino acid residues. The R groups (the variant groups) of the polypeptide protrude out from the α-helix chain. In the β-pleated sheet, the “pleats” are formed by hydrogen bonding between atoms on the backbone of the polypeptide chain. The R groups are attached to the carbons and extend above and below the folds of the pleat. The pleated segments align parallel or antiparallel to each other, and hydrogen bonds form between the partially positive nitrogen atom in the amino group and the partially negative oxygen atom in the carbonyl group of the peptide backbone. The α-helix and β-pleated sheet structures are found in most globular and fibrous proteins and they play an important structural role.

Tertiary Structure

The unique three-dimensional structure of a polypeptide is its tertiary structure (Figure 3.29). This structure is in part due to chemical interactions at work on the polypeptide chain. Primarily, the interactions among R groups creates the complex three-dimensional tertiary structure of a protein. The nature of the R groups found in the amino acids involved can counteract the formation of the hydrogen bonds described for standard secondary structures. For example, R groups with like charges are repelled by each other and those with unlike charges are attracted to each other (ionic bonds). When protein folding takes place, the hydrophobic R groups of nonpolar amino acids lay in the interior of the protein, whereas the hydrophilic R groups lay on the outside. The former types of interactions are also known as hydrophobic interactions. Interaction between cysteine side chains forms disulfide linkages in the presence of oxygen, the only covalent bond forming during protein folding.

All of these interactions, weak and strong, determine the final three-dimensional shape of the protein. When a protein loses its three-dimensional shape, it may no longer be functional.

Quaternary Structure

In nature, some proteins are formed from several polypeptides, also known as subunits, and the interaction of these subunits forms the quaternary structure . Weak interactions between the subunits help to stabilize the overall structure. For example, insulin (a globular protein) has a combination of hydrogen bonds and disulfide bonds that cause it to be mostly clumped into a ball shape. Insulin starts out as a single polypeptide and loses some internal sequences in the presence of post-translational modification after the formation of the disulfide linkages that hold the remaining chains together. Silk (a fibrous protein), however, has a β-pleated sheet structure that is the result of hydrogen bonding between different chains.

The four levels of protein structure (primary, secondary, tertiary, and quaternary) are illustrated in Figure 3.30.

Denaturation and Protein Folding

Each protein has its own unique sequence and shape that are held together by chemical interactions. If the protein is subject to changes in temperature, pH, or exposure to chemicals, the protein structure may change, losing its shape without losing its primary sequence in what is known as denaturation. Denaturation is often reversible because the primary structure of the polypeptide is conserved in the process if the denaturing agent is removed, allowing the protein to resume its function. Sometimes denaturation is irreversible, leading to loss of function. One example of irreversible protein denaturation is when an egg is fried. The albumin protein in the liquid egg white is denatured when placed in a hot pan. Not all proteins are denatured at high temperatures for instance, bacteria that survive in hot springs have proteins that function at temperatures close to boiling. The stomach is also very acidic, has a low pH, and denatures proteins as part of the digestion process however, the digestive enzymes of the stomach retain their activity under these conditions.

Protein folding is critical to its function. It was originally thought that the proteins themselves were responsible for the folding process. Only recently was it found that often they receive assistance in the folding process from protein helpers known as chaperones (or chaperonins) that associate with the target protein during the folding process. They act by preventing aggregation of polypeptides that make up the complete protein structure, and they disassociate from the protein once the target protein is folded.

Link to Learning

For an additional perspective on proteins, view this animation called “Biomolecules: The Proteins.”

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Research output : Contribution to journal › Article › peer-review

T1 - Solubility and digestibility of milk proteins in different forms of infant formulas

N2 - Heat processing is necessary to extend the shelf-life of commercially manufactured infant formulas. Heat treatment causes protein denaturation and may thereby affect protein digestibility. Currently, there are several fundamentally different technologies to produce infant formulas such as sterilization which used for production of liquid formulas, and spray-drying which is used for production of powdered formulas. Infant formula (liquid concentrated, powdered, ready-to-feed), prepared according to the manufacturers' instructions were centrifuged at 14,000 g at 4°C for 1 h. Fractions [lipid layer, soluble fraction (whey) and pellet (casein)] were separated. Total N and N in fractions were determined by micro-Kjeldahl analysis and non-protein nitrogen (NPN) after precipitation of proteins. Protein solubility was determined after centrifugation and ranged from 5790%. To simulate the infant gut, in vitro digestion was used. Formulas were adjusted to pH 4.5 with 0.1 N HC1 and pepsin was added (lmg/ml). Pancreatin (0.4 g/100 ml of 0. l M NaHCO3) was added to each sample, which was then incubated at 37°C. Digested formulas were immediately placed in boiling water for 4 min. to inactivate enzymes. True protein content ranged from 12.5-17.5 g/L. Protein digestibility calculated as (NPN after digestionNPN before digestion) x 6.25 x 100/true protein ranged from 49-68%. Protein digestibility was generally highest for powdered formula, while it was slightly higher from liquid concentrate than from ready-to-feed formula. These results suggest that protein utilization by infants may be affected by the type of processing used.

AB - Heat processing is necessary to extend the shelf-life of commercially manufactured infant formulas. Heat treatment causes protein denaturation and may thereby affect protein digestibility. Currently, there are several fundamentally different technologies to produce infant formulas such as sterilization which used for production of liquid formulas, and spray-drying which is used for production of powdered formulas. Infant formula (liquid concentrated, powdered, ready-to-feed), prepared according to the manufacturers' instructions were centrifuged at 14,000 g at 4°C for 1 h. Fractions [lipid layer, soluble fraction (whey) and pellet (casein)] were separated. Total N and N in fractions were determined by micro-Kjeldahl analysis and non-protein nitrogen (NPN) after precipitation of proteins. Protein solubility was determined after centrifugation and ranged from 5790%. To simulate the infant gut, in vitro digestion was used. Formulas were adjusted to pH 4.5 with 0.1 N HC1 and pepsin was added (lmg/ml). Pancreatin (0.4 g/100 ml of 0. l M NaHCO3) was added to each sample, which was then incubated at 37°C. Digested formulas were immediately placed in boiling water for 4 min. to inactivate enzymes. True protein content ranged from 12.5-17.5 g/L. Protein digestibility calculated as (NPN after digestionNPN before digestion) x 6.25 x 100/true protein ranged from 49-68%. Protein digestibility was generally highest for powdered formula, while it was slightly higher from liquid concentrate than from ready-to-feed formula. These results suggest that protein utilization by infants may be affected by the type of processing used.

Proteins are large molecules found in our bodies and food, consisting of many smaller components called amino acids. Proteins have the properties they do because of the shape and arrangement of their amino acids. A weak bond, known as a hydrogen bond, forms between a hydrogen atom and an oxygen atom in the amino acids. This gives the protein its shape.

What is denaturing and how does it happen?
A protein becomes denatured when its normal shape gets deformed because some of the hydrogen bonds are broken. Weak hydrogen bonds break when too much heat is applied or when they are exposed to an acid (like citric acid from lemon juice). As proteins deform or unravel parts of structure that were hidden away get exposed and form bonds with other protein molecules, so they coagulate (stick together) and become insoluble in water. Curing salmon using lemon and lime juice (eg. to make a gravadlax or ceviche) is an example of protein acid denaturation.

Washing the plate

The two most commonly used wash buffers in ELISA applications are Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20. To wash a plate, wells should be repeatedly filled and emptied by either aspiration or plate inversion (i.e., dumping and flicking solution into a suitable receptacle). Generally at least 3 x 5 minute washes should be applied after the incubation of coating antibody, sample, and detection antibody 6 x 5 minute washes should be given after incubation with the enzyme conjugate. It is not necessary to wash after the blocking step, although this is not detrimental to the assay. Wash buffers should be used in sufficient volumes to completely wash the wells. For example, 400 μL is generally used for each well of a 96-well plate.

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Select products

Antibodies are a key part of any ELISA workflow. For an antibody to work successfully in ELISA, it should react specifically with the antigen but not cross-react with any other component of the assay. Not all antibodies can be used successfully in ELISA applications, individual antibodies must be evaluated. For sandwich assays, where two different antibodies are required, it is essential that the two antibodies react with different epitopes on the antigen or an epitope that appears several times on the antigen.

For example, if the antigen is immobilized on the plate through the capture antibody, then the detection antibody must be able to interact with its own epitope without steric hindrance from the first antibody. In ELISA applications where a secondary antibody is used as part of the detection complex it is also essential that the capture and detection antibodies be raised in different animal species so that the secondary antibody does not react with the coating antibody. Antibodies that work well together are generally known as “matched pairs”. Most commercially-available ELISA kits use validated matched antibodies pairs, and these pairs are often available for purchase individually or as “Antibody Pair Kits”.

In addition, antibody concentrations should be considered when setting up a new ELISA. Each antibody being used will require optimization. The optimal range is partially determined by the form and origin of the antibody and also by the substrate used for signal generation. When diluting antibodies, detection antibody and enzyme conjugate, working solutions should be prepared in blocking solution to reduce non-specific interactions. For recommended coating and detection antibody concentrations see below.

Table 2. Recommended concentration ranges for coating and detection antibodies for ELISA optimization. The use of non-purified antibodies will work but may result in higher background. It is generally recommended to use affinity purified antibodies for optimal signal-to-noise ratio. Concentrations are guidelines only for best results optimize each component individually.

Watch the video: Salting in and salting out (May 2022).