CHAPTER 2 Estimation of Proteins and Amino acids

CHAPTER 2 Estimation of Proteins and Amino acids Suresh Kumar and Sweta Kumari Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi-110012, India. Introduction Proteins are not only the bodybuilding constituent of an organism but also the catalysts/ enzymes for biochemical reactions. Plant cells differ from animal cells in that they have a rigid cell wall, and often have vacuoles that contain secondary metabolites (especially phenolics & polyphenolics), organic acids and proteinases. When the plant tissues are ground into fine powder, the vacuoles are ruptured releasing the contents that may degrade, inactivate, modify and/or precipitate the proteins. Therefore, special care is required to protect the proteins. Though proteins are extracted in the laboratories for a variety of reasons, there are two main purposes for their extraction from plant tissues: (i) to estimate total protein content in crude plant extract, (ii) to purify an enzyme or a protein. The method that should be used for protein extraction would depend on the purpose, type of the plant sample, and the enzyme or protein to be purified and estimated. The more we know about the characteristic features of the protein/enzyme of interest, it is easier to choose the method of extraction. In general, younger plants tissues are easier to grind into fine powder, and often contain fewer interfering (inactivator, phenolics, proteinases etc.) compounds. There are several biochemical parameters that must be considered to improve stability, protection, recovery, and yield of the protein(s) to be isolated/purified. Some of the parameters are briefly described below. The critical parameters Extraction buffer: As the content of vacuoles is quite acidic, sufficient buffering capacity of the medium must be ensured to avoid drastic change in the pH from the acceptable range for the protein of interest. Buffer maintains pH of the medium as well as ionic-strength to stabilize the proteins/enzymes. MOPS (N-morpholino propane sulfonic acid, pH 7.0) buffer is the most commonly used buffer for extraction of proteins; however, other buffers including HEPES (N-2-hydroxyethyl piperazine -2-ethane sulfonic acid) and MES [phosphate, or 2-(Nmorpholino)-ethanesulfonic acid] can also be used depending on the purpose of extraction of proteins and the needs to avoid a particular buffer. Certain proteins show optimal solubility at higher pH; therefore, Tris base may be included to raise pH of the extraction buffer. Moreover, different proteins are soluble at different pH, so different pH of the extraction buffer may be utilized to extract a different set of proteins. The volume of extraction buffer to be used per unit weight of plant sample depends on the type of tissue and the purpose of extraction of the proteins. Generally, better extraction of proteins is expected with a higher ratio (for example 10:1) of buffer (volume) to the plant sample (weight). However, for extractions of a larger amount of proteins this ratio may result in too large volume to work within the subsequent steps. If the ration of extraction buffer to plant tissue is not sufficient enough, this may cause a change in pH of the extract and inactivation of the extracted proteins/enzymes. The buffer may also contain 10%−20% glycerol to ensure the stability of

certain unstable proteins. Some proteins (e.g. certain seed storage proteins) require ionic interactions for solubility, so they are soluble only in the presence of higher (0.1 to 1 M) salt concentrations. Moreover, the use of salt requires caution because of its disruptive effect in the downstream processes (e.g. separation of proteins by isoelectric focusing). Antioxidants: Antioxidants are added in the extraction buffer to maintain reduced state of free sulfhydryl groups of enzymes and to reduce oxidation of other components, such as phenolics. The antioxidants which are commonly used include DTT (2 mM or more), β-mercaptoethanol (5−10 mM), or ascorbic acid (5−10 mM). Under certain circumstances, even stronger reducing agent (such as sodium dithionite) and more than one reducing agents (e.g. DTT and β-mercaptoethanol) can also be used. Antioxidants also reduce the activity of polyphenol oxidases which synthesize polyphenols from phenolic compounds upon tissue disruption in the presence of oxygen. Detergents: Detergent [e.g. Sodium Dodecyl Sulfate (SDS)] is used in the extraction buffer to disrupt cell membranes. Under certain circumstances, they are useful for maximizing the yield of soluble proteins, especially when the low ratio of extraction buffer to tissue is used. Other detergents [e.g. Tween 80 (0.1%−1%), Triton X-100 (0.1%−1%)] can also be used for differential extraction. In general, initial testing is required to determine the optimal combination and concentration of the detergents to be used. Protective agents: Protective agents are generally required in the extraction buffer to prevent polyphenolic compounds [e.g. tannins (water-soluble polyphenols of varying molecular weight)] to bind and inactivate proteins. Generally, Polyethylene glycol (PEG 6000), insoluble PVPP or soluble PVP (polyvinyl pyrrolidone) are used in most of the applications which bind polyphenols best at the pH < 7. PVPP is used @ 0.5−5% (w/v) which requires to be hydrated for 1 hour in the extraction buffer before use, and it can be removed from the medium by filtration and/or centrifugation. Proteinase inhibitors: Proteinases are released from vacuoles upon disruption of the cells; therefore, use of proteinase inhibitors is essential for minimizing the activities of proteinase and to control the degradation of proteins. Cysteine proteinases are most common in plant tissues but metalloserine, and -aspartic proteinases have also been reported in different tissues. Some proteinase inhibitors are irreversible and chemically modify their targets (for example, phenylmethylsulfonyl fluoride, PMSF), whereas others are tight-binding (for example, bovine pancreatic trypsin inhibitor, BPTI). On the other hand, some proteinase activity may be recovered in later steps if the proteinase inhibitor is not present. Generally, PMSF (1 mM) and BPTI (1 µM) are used against serine proteinases. Since PMSF is toxic and rapidly hydrolyzed, it must be used carefully and should be added to the extraction buffer just before tissue disruption. EDTA (1−10 mM) is used against metalloproteinases, while transepoxysuccinyl-L-leucyl-amido-(4-guanidino) butane, (10 µM) is effective against cysteine proteinases. Antipain and leupeptin (10 µM) have activity against all serine and cysteine class proteinases. Commercial proteinase inhibitors (available in the form of the tablet from Roche, or solution from Sigma) containing a cocktail of these inhibitors can be purchased from the supplier. It must be remembered that the proteinase inhibitor tablet is slow to dissolve; therefore, it should be added to the extraction buffer about 30 min before using the buffer for protein extraction.

Tissue types: The type of tissue used for protein extraction determines the method which should be used and the yield of protein which can be obtained. In leaf, protein concentration is high (1%−4%), while stem tissues may have a lower amount of proteins. Therefore, the use of a higher extraction buffer ratio will lead to very low concentration of proteins in the extract. Seeds (usually having