Lysozyme Enzymes: Purification and Assaying

Lysozyme Enzymes: Purification and Assaying Abstract This lab investigates the purification and assaying of lysozyme using size exclusion chromatography and Bradford protein assay. Micrococcus Lysodeikticus is employed in this lab as the substrate for lysozyme and its enzymatic activities are observed under various pH and substrate concentrations for determining the optimal conditions for lysozyme activity. The highest lysozyme activity was observed at a substrate concentration of 0.4 mg/mL and at an optimum phosphate buffer pH of 7. These optimal conditions were set as standard conditions for assaying the purified fractions for lysozyme activity and for the protein assay. A solution of egg white is used as the source for lysozyme and it is purified using size exclusion chromatography with column Sephadex G-50 that has a fractionation range from 1,500 to 30,000 daltons. Size exclusion chromatography provides purification of lysozyme, however about 64% of its initial yield is lost in the process of purification through the beads. The hi ghest lysozyme activity was observed for fraction # 12 indicating a structural mass range of 15,000da-13,500da for lysozyme. The protein assay indicated a significant concentration of protein in the neighbouring fractions of purified lysozyme, however the exact concentration of lysozyme in albumen remain inconclusive. Introduction Lysozyme is a catalytic enzyme that digests bacterial cell wall and is found in significant amounts in egg whites. Egg white, also known as albumen, is the cytoplasm of the unfertilized egg cell, which consists of approximately 40 different proteins dissolved in water (Lee-Huang et al. 1999). The main proteins in albumen are ovalbumin, ovotransferrin, ovomucoid, globulins, lysozyme, ovomucin, avidin, etc (Lee-Huang et al. 1999). Lysozyme comprises about 3.5 % of the total protein weight in albumen (Lee-Huang et al. 1999) and thus a solution of egg white was used as the source of lysozyme in this lab. The main objective of this lab is to purify lysozyme using size exclusion chromatography for examining its structural properties and concentration in albumen. In this experiment lysozyme is extracted and purified from albumen using size exclusion chromatography. The stationary phase in the column consists of a porous cross linked gel matrix of Sephadex G-50 with a fractionation range of 1,500da to 30,000da. Separation and purification by size exclusion chromatography is based on molecular size i.e. larger molecules elute first with the mobile phase while the smaller molecules get trapped within the beads and elute last (Lodish et al. 2000). The different proteins present in albumen should elute at different fractions due to their varying molecular sizes leading to the purification of lysozyme which has a literature structural mass of 14,400da. It is hypothesized that since lysozyme is a small molecule in comparison to the fractionation range of the Sehadex G-50 coloumn, a significant amount of lysozyme is likely to be trapped or retarded by the resin beads resulting in a low yield. Substrate binding is used as a powerful tool in this experiment where the enzymatic reactions are used for detecting the presence of lysozyme. The structural composition of lysozyme consists of 129 amino acid residues folded into a compact globular structure with a cleft for substrate binding (Berg et al. 2002). When a substrate binds to the cleft, it hydrolyzes the peptidoglycan polysaccharide found in many bacterial cell walls, resulting in the osmotic lyses of the cell (Berg et al. 2002). Gram positive bacteria are more susceptible to the effects lysozyme due to their peptidoglycan cell wall being exposed to the extracellular environment (Lee-Huang et al. 1999). However, gram negative bacteria are less vulnerable to the presence of lysozyme due to their thin layer of peptidoglycan shielded by the outer membrane of lipopolysaccharide (Lee-Huang et al. 1999). In this lab, the gram positive bacteria of Micrococcus Lysodeikticus is used as a substrate for detecting the enzymatic activ ities of lysozyme. The Bradford assay on the other hand is used to estimate the concentration of lysozyme with respect to other major proteins present in albumen. Lysozyme has great research importance since it possesses the capability to lyse gram positive bacteria. Lysozyme, like most of the other biomolecules are not found in nature in its isolated form and this lab investigates one of the most simplest methods for extracting and purifying lysozyme from albumen. Materials and Methods A solution of egg white diluted to  ¼ with 0.1 M phosphate buffer pH 7 and filtered though glass wool is used as the source for lysozyme (Laboratory Manual. 2007). The solution is put through size exclusion chromatography with G-50 Sephadex column (fractionation range of 1,500-30,000 da) to produce 24 test tubes of equal egg white fractionations of 0.75 mL (Laboratory Manual. 2007). Numerous assays are conducted with varying pH and micrococcus (substrate) concentration to determine the optimal conditions for the highest enzymatic activity of lysozyme. After the collected column fractions and prepared egg white solution were left in the lab for two weeks, the odd numbered test tubes were assayed for lysozyme activity and the even numbered test tubes were assayed for protein at optimal pH. Refer to York University Department of Biology Laboratory Manual Summer 2008, SC/Biol 2020 Cell Biology and Biochemistry Pages 54-57 for a more detailed procedure of the lab. Also refer to the attached flow sheets for a thorough step by step procedure for this lab. Results The addition of micrococcus to a solution of lysozyme results in the rapid decrease in its optical density value due to its enzymatic reactions. Various assays are conducted in this lab to examine various properties of lysozyme. The substrate concentration assay indicated 0.4 mg/mL of micrococcus to be the optimal substrate concentration for lysozyme as it resulted in the highest enzymatic activity of 250 units. The pH assay on the other hand indicated pH 7 to be the ideal pH for the phosphate buffer as it resulted in the high lysozyme activity of 300 units. These observations led us to set 0.4 mg/mL micrococcus and buffer pH of 7 as standard conditions for assaying the size exclusion column fractionations for protein activity. When assaying the odd numbered fractionations for lysozyme activity, fraction # 15 reached the highest enzymatic activity of 900 units specifying the presence of concentrated lysozyme in that fraction of egg white. The neighbouring fractions (#14 and #16) show ed significant protein concentrations of 4.4 mg/mL and 1.6 mg/mL when assayed however the highest protein concentration as observed in fraction # 12 which indicated a protein concentration outside the standard curve range. Upon dilution, the protein concentration of fraction # 12 was calculated to be 9mg/mL. Based on the results, a protein fold of 1.36 was calculated and the results showed a high lysozyme yield loss of about 64%. Calculations: Sample calculation of Micrococcus dilution: Target: 3 ml of 0.4 mg/ml Micrococcus C1V1 = C2V2 (10 mg/ml)(x ml) = (0.4 mg/ml)(3 ml) X = 0.12 ml of Micrococcus 3 ml 0.12 ml = 2.88 ml Therefore, 0.12 ml of Micrococcus and 2.88 ml of Phosphate buffer will be required Sample calculation of total protein in fraction # 15: protein concentration of 0.52 mg/mL Volume in fraction: 0.1 mL Total protein = 0.52mg/mL X 0.1mL = 0.052 mg Sample calculation of Total Enzyme Activity in fraction # 15 Activity = ΔOD x 1min/0.001 Activity = (0.4) x 1min/0.001 Activity = 400 units Sample calculation of Specific Activity for fraction # 15: Enzyme activity of column fraction 15 = 90 units Total protein = 0.052 mg Specific Activity = Enzyme Activity/Total protein Specific Activity = 400 units/( 0.052 mg) Specific Activity = 7692 units/mg protein Sample calculation of initial specific activity of the egg white at 0.3 mg/ml substrate: Specific Activity = Enzyme Activity/Total Protein Specific Activity = 90 units/(4.5 mg/ml x 0.1 ml) Specific Activity = 200 units/ mg protein Calculation of Fold Purification: Fold Purification = Specific Activity of fraction/Specific Activity of egg white Fold Purification = (7692 units/ mg protein) / (200 units/ mg protein) = 38.46 Discussion This lab experiment examines the purification and assaying of egg white lysozyme. Lysozyme is both a protein and an enzyme that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan (Lodish et al. 2000). It is found in abundant quantities in albumen (egg whites) where it protects the egg embryo from bacterial invasion. Several assays were conducted in this lab experiment and each assay demonstrated a significant property of lysozyme. Enzymes have optimal conditions at which it functions most effectively and it is important that enzymes are studied under optimal conditions for the most accurate results and observations. The substrate concentration assay and pH assay were employed in this experiment to determine the optimal conditions for lysozyme since its enzymatic activity varies with substrate concentration and buffer pH. Appropriate substrate concentration is significant for an enzymatic reaction because a high substrate concentration might outnumber the available active sites on lysozyme while a low substrate concentration will leave vacant binding sites on the lysozyme. This lab proved 0.4 mg/mL of Micrococcus to be the optimal substrate concentration for lysozyme with a high enzymatic activity of 250 units. The determination of the optimal pH at which the substrate-enzyme binding is carried out most efficiently is another important aspect of an enzymatic reaction. An environment too acidic or basic could ca use hindrance for the substrate-enzyme binding and thus result in low lysozyme activity. The phosphate buffer pH assay in this lab proved pH 7 to be the optimal pH at which the enzymatic activities of lysozyme are carried out most efficiently with an enzymatic activity of 300 units. Size exclusion chromatography is used in this experiment for extracting and purifying lysozyme from the mixture of approximately 40 proteins that is present in albumen. Other proteins that are present in significant quantities in egg whites are ovalbumin (66 kDa), ovotransferrin (77.8 kDa), ovomucoid (28 kDa), ovomucin, avidin (18kDa), etc. however none of these proteins share the same molecular mass as lysozyme which has a literature molecular weight of 14.5 kDa (Lee-Huang et al. 1999). This unique mass distinction between the proteins present in albumen allows size exclusion chromatography to be an effective method in purifying lysozyme as its separation method is solely based on molecular mass difference. Specific selection of resin for the column is another important factor in protein purification as the porosity and fractionation range of the column should be focused on the protein being purified. This experiment uses the Sephadex G-50 column for protein purification. The stationary phase of Sephadex G-50, has a bead matrix with a well defined pore size for separating proteins within the fractionation range of 1,500da 30,000da. Sephadex G-50 is well suited for lysozyme purification because lysozyme has a literature molecular mass of 14,400da (Lee-Huang et al. 1999) which falls midway between the column fractionation range. Purification of lysozyme however would come with the cost of obtaining low lysozyme yields. During purification, the larger molecules elute first while the smaller molecules like lysozyme travel through the beads and elute last. This affects the yield of lysozyme as some of its initial mass gets trapped within the gel matrix beads during separation while another small fraction of the initial yield is lost due to the retardation of the lysozyme during the in and out diffusion of the protein from the beads in the matrix (Laboratory Manual. 2007). Thus for a method like size exclusion chromatography, I would predict very low yields. The prediction was proved to be true as the data collected from the experiment demonstrated a low yield of 36%. Thus even though the method of size exclusion chromatography is effective in purifying lysozyme, it comes with the disadvantage of low yield. Micrococcus, the substrate for lysozyme in this experiment, is a gram positive bacteria with an exposed peptidoglycan cell wall (Lee-Huang et al. 1999). Like any other enzymes, lysozyme is very specific about its substrate and the ability of lysozyme to bind to micrococcus and lyse the cell allows us to follow its enzymatic activity through the decrease in optical density detected on the spectrometer. Lysozyme activity assay demonstrated a peak for the highest enzymatic activity at fraction # 15. The peak represents the single protein species of lysozyme because micrococcus can only be digested by lysozyme and no other albumen proteins. Therefore all activities observed for lysozyme assay is due to the presence of lysozyme binding and hydrolyzing the peptidoglycan in the cellular walls of micrococcus. Micrococcus is thus the ideal substrate for this experiment however it also poses some disadvantages as well due to its biohazardous nature. Micrococcus needs to be handled with extra c aution and is to be discarded of appropriately. The lysozyme assay showed the highest specific activity of 900 units in fraction # 15. This indicated that fraction # 15 contained purified lysozyme. Considering the fractionation range of 1,500 30,000 da of the chromatography, and the elusion of a fixed volume into 24 separate test tubes, an estimation of the molecular weight range for fraction # 15 could be made around 15,000 da 13,500 da, a range that covers the literature molecular mass of lysozyme, 14,400 da. Thus, our detection for the fraction of egg white containing purified lysozyme was pretty accurate. The Bradford protein assay was used in this lab to examine the protein concentration of lysozyme with reference to the other proteins present in albumen. Theoretically lysozyme comprises about 3.5% of the total protein mass in albumen (Canà §ado et al. 2007). Even though it is a significant amount, there are other proteins comprising a higher concentration in albumen. The odd fractionations closest to #15 show fair concentrations of protein with # 14 showing a concentration of 4.4 mg/mL and # 15 showing 1.6 mg/mL. In order to get an accurate reading for the lysozyme protein concentration, fraction # 15 would have to be directly assayed instead of its neighbouring fractions. Thus the actual lyoszyme concentration in albumen remains inconclusive. The highest protein peak was observed for fraction # 12 with a protein concentration that went beyond the standard curve range. Upon dilution, the protein concentration of # 12 was determined to be approximately 9 mg/mL. This indicates that a protein that eluted in fraction # 12 is the most concentrated in albumen. Specific activity is defined as the enzyme activity over total amount of protein (Laboratory Manual. 2007). The specific activity of 900 units/mg protein for fraction 15 and the specific activity of the initial egg white solution of 666.66 a fold purification of 1.35(Refer to calculations). The yield of lysozyme after purification was fairly low indicating a high percent of loss. This loss was due to a portion of lysozyme being trapped within the beads of the matrix during purification and another small portion being distorted during in and out diffusion of lysozyme (Laboratory Manual. 2007). The 64% loss in enzyme yield was worth the increase in purity because purifying the enzyme enabled us to estimate its structural mass and protein concentration in albumen. Sources of error in this lab were tried to be kept at its minimal level however there could still be some errors that might have deviated the results slightly. Lysozyme activity was measured by detecting the difference in optical density on a spectrophotometer in a time period of 1 minute. Incorrect readings of the optical density caused by fingerprints/other residue on the test tube surface or reading the incorrect absorbance at an earlier or later time period could have been a major source of error as this lab is dependent on the accuracy of the optical density readings. Other sources of error could be dilution errors as some of the dilution require very minute amounts and pipetting the small amounts with the pipette provided for this lab accurately is very challenging. New researches in the field of biochemistry help expand our knowledge about cell and molecular processes and thus research interest in the unique enzyme of lysozyme is of no exception. It was previously believed that lysozyme was used primarily as a constitutive defense against bacterial pathogens but recent research indicate that in certain species in the animal kingdom the structure of lysozyme is different and the structural difference enables lysozyme to incorporate other useful functions such as digesting bacteria for nutrition (Canà §ado, et al., 2007). Research has also shed light on urinary lysozyme C showing that a combination of urinary lysozyme C with certain RNases can be used to combat HIV-1 (Lodish et al. 2000). Other researches on lysozyme conducted by Lee-Huang et al. found that lysozyme from chicken egg white, human milk and human neutrophils combined with RNase A from bovine pancreas display activity against HIV-1 (Lee-Huang, et al., 1999). These are significant di scoveries that not only broaden our knowledge in biochemistry but also define possible cures for HIV in the future. Conclusion Lysozyme is a widely distributed enzyme in the animal kingdom that lyse bacterial cells to protect organisms from bacterial invasion and this lab demonstrated some of the important characteristics of this unique enzyme. Micrococcus proved to be an ideal substrate for observing lysozyme activity due to its gram positive nature illustrated by its exposed peptidoglycan cell wall, the cleavage target of lysozyme. The substrate concentration assay and pH assay demonstrated how lysozyme is at its peak enzymatic activity at the optimal substrate concentration of 0.4 mg/mL and at a buffer pH of 7. Even though the exact protein concentration of lysozyme in albumen remains inconclusive, the neighbouring fractions assayed for protein concentration provided an estimate that lysozyme is present in significant amounts in egg white in comparison to the other albumen proteins. Even though lysozyme was successfully purified using size exclusion chromatography with Sephadex G-50 column, its purificati on resulted in the loss of lysozyme yield. The column beads trapped and retarded about 64% of the total lysozyme resulting in low yields. Overall this was lab was well engineered to demonstrate how size exclusion chromatography can be used for purification based on molecular mass and how the unique activities of a certain enzyme with its specific substrate can be used to determine the purified fraction that contain the certain enzyme. Needs of the Elderly: A Case Study Needs of the Elderly: A Case Study Introduction We live in an ageing world and Australia is not an exception. In 1901 only 4% of Australians were 65 and older, by 2016 that figure increased to 15.3% and is estimated to rise to 23% by 2041 (Australian Bureau of Statistics, 2016, Australian Human Rights Commission (AHRC), 2012). The United Nations (UN) principles of ageing, supported by the Australian Government, recognises the rights of all older persons, focusing in areas such as: independence; participation; care; self-fulfilment and dignity (Australian Association of Social Workers (AASW), 2013), but are we doing enough to protect these rights and ensuring our older Australians are not left disadvantaged? This essay looks at the case of Hank, retrenched from his lifelong employer at the age of 62 with future employment prospects bleak. It looks at factors through the life course that effect Hank in his older years and discovers how Social Workers can respond to this demographic to assist them in continuing to fulfil a satisfying life. Key Social, Psychological and Physical Needs As a social worker, it is important to have an understanding of the key social, psychological and physical needs of the client plus any issues that may be present. In this way, a holistic and specific needs assessment can be made that identifies how a client situation affects his needs moving forward (Tanner Harris,2008). Hank was retrenched from his workplace and is having difficulty finding a new job. The jobs he is experienced in are becoming unsuitable due to the onset of arthritis and without formal training, his employment prospects are bleak. There have been many studies linking unemployment with lower levels of well-being, and higher levels of distress, self-doubt and dissatisfaction (VanDerMeer,2014). Being unemployed can be detrimental to ones needs, not only because of lesser income but also due to the effects on psychological and social factors (VanDerMeer,2014). Societal norms and expectations also plays an important role in well-being, being employed helps us to feel w e are conforming to those values and norms (Thompson,2016, VanDerMeer,2014). Mens social identity is often centred around their work and although as a society we are shifting in our view of men being the sole bread winner, for Hanks cohorts, his role as financial provider gave him a sense of pride and sense of identity. Hanks friends, his social group, have remained at work, this puts added stress on Hanks wellbeing, people who are unemployed are better able to cope when others around them are in the same situation (Clark,2003). Human Rights and Legislation Hank, like all Australians, have a universal right to employment regardless of factors such as culture, gender, age and disability (Ife,2012). The Australian Government, through the Australian Human Rights Commission, has agreed to uphold the UN covenants on human rights. Although there is no international covenant in the area of ageing, the Universal Declaration Of Human Rights (1948, art.23) and the International Covenant On Economic, Social And Cultural Rights (1966, p.3,art.6) states that everyone has the right to work and be paid without discrimination, and that protections should be in place against unemployment. Federal legislation in place to uphold Australian Human Rights include the Australian Human Rights Commission Act 1986 and Age Discrimination Act 1992. In Victoria, workers rights are protected through the Equal Opportunity Act 2010, which includes Age and Disability. Adhering to such rights gives all people a sense of human dignity, a value held by the Australian Asso ciation of Social Workers (AASW) in its code of ethics (2010). These protections should be an advantage to Hank but unfortunately, in reality age discrimination, modernisation and globalisation often make it difficult to apply. Impact of Structural and historical Factors Although ageing happens to us all regardless of race, culture, gender or class, these structural factors, economic and social policies, plus experiences throughout the life course, often have a significant influence on later life (Hughes Heycox,2010). That influence, restrained by economic and social policies, has the ability to limit choices and opportunities that impact on ageing (Bengtson DeLiema,2016). Australias increasing population and its effect on the dependency ratio (the portion of population who are too young or old to work by those of working age), will also have an impact on factors such as housing, medicare, Centrelink, superannuation and age care. If Hank turns 63 in 2017, he was born in 1954, the baby boom generation. The year of birth is not only important in determining Hanks generational cohorts and possible life course experiences, but also in determining his eligibility for services such as the aged pension and superannuation release. The baby boomers were so called due to the raise in babies born during and post world-war II 1943 -1964 (Tolbize,2008). According to Tolbize (2008, p.2), it was an era of prosperity and optimism and bolstered by the sense that they are a special generation capable of changing the world, have equated work with self-worth, contribution and personal fulfillment. Generalising on the typical situation of this generation, Hank and his cohorts would have had a strong sense of work and sacrifice. They believed in loyalty to one employer with success being gained through step-by-step promotion, of paying their dues (Tolbize,2008). Hank was born in the pre-globalisation era where people could more e asily get a job without the need for a university or formal trade education (Phillipson,2009). Hank chose to follow the path of his father, leave school at 16 and start work as a machine operator. He expected, just as his father did, to be in his job for the rest of his working life. However, the recent globalisation of the Australian economy and technological advances, are often reflected in retrenchment of lower skilled workers like Hank and the creation of structural unemployment where unemployment results from shifts in the economy and by factors such as technological advances, making lower-skilled jobs obsolete (Business Council of Australia (BCA),2004, Klehe et.al,2012). Government measures are normally in place to provide assistance in the form of retraining in such situations. For example, when industries such as the car manufacturing, the government purposed other needed industries for retraining such as, ironically, aged care. For older workers, such as Hank, the offer i s often refused. Hanks self-devaluing attitude based on the ageist stereotype of you cant teach an old dog new tricks is often a barrier to changes in ones employment sector. Based on his year of birth, Hank will be eligible for the aged pension at age 66. Since April 1909, when Australia commenced the Invalid and Old Age Pensions Act 1908 for people aged 65, Australias retirement, pension and superannuation policies have become an important factor in the ageing experience. Since then, Australia has made changes to encourage Australians to be more self-sufficient in their retirement, therefore removing the burden of financial assistance from the government. 1915, saw the Income Tax Assessment Act 1915 allowing employers to make contributions on behalf of its employees for superannuation. In 1972 only 32% of Australians where covered by superannuation. In the 80s, the Federal government established policies around superannuation and taxation, this, plus the introduction of superannuation contribution by employers, increased the number of employees covered by Superannuation in 1990 to 64%. In 1992 the superannuation guarantee was implemented, employers were required to contribute to their employees superannuation fund. The amount of contribution progressively increased from 3% to 9% in 2002. In July 2017, the qualifying age for the age pension will increase by 6months every two years until it reaches 67 years of age in 2024 (Parliament of Australia,2010). The impact of government policy in Hanks situation means, that although he will not be eligible for the aged pension until 66, he is eligible to access his superannuation (Australian Department of Human Services (DHS),2017). The Department of Human Services through Centrelink is responsible for income support such as newstart, however in Hanks case, it is unclear if he is eligible for newstart based on his wifes full-time income. Victorian Skills Gateway, which is part of the Victorian Government Department Education and Training (DET) offices help with retraining and job search (DET,2017). Knowing the importance of older workers in the workforce, the government restart program offers incentives for employers to hire people over 50 (DET,2017). They are also looking at reforming structural barriers such as workers compensation, income protection and professional licensing all factors that affect employability of older workers. This is with the hope, that by removing these barriers, will remove negative perceptions of employing older workers (AHRC,2012). Theories of Ageing In the last few decades there has seen the development of many theories on ageing that allow us a better understanding of an older persons world. As a social worker, we can draw upon theories to gain clarity in understanding aspects of the human condition (Tanner Harris,2008, Hughes Heycox,2010). Theory can also enable us to find appropriate approaches to dealing with clients based on their specific needs and situations (Tanner Harris,2008). One of the pioneer theories on ageing, which draw popularity in the 60s through to the mid-seventies is disengagement theory. Disengagement theory, as proposed by Cummings and Henry (1961) states that ageing involves the gradual and inevitable withdrawal of a person from interacting with society. It supposes that this inevitable withdrawal is mutually beneficial, as it sees the transfer of power from the old members of society to the young. According to this theory, to successfully age means a reduction in activity levels and interactions until all activities cease in preparation for death (Hughes Heycox,2010). This enables the young a greater opportunity to become more active in contributing to society, thereby keeping society in balance (Victor,2005). Although Hank did not voluntarily leave the workforce, according to this theory, his retrenchment has set off his path to disengagement. We see this through his discouragement with not finding another job and therefore dropping out o f the job search and isolating himself from his friends. Proponents of this theory, would see this as natural progression for Hank Hank is on natural course leading to the remainder of his life. The problem with this theory in its original form however, is that it doesnt consider social processes and structures that are in place, that may hinder a person such as Hanks ability to engage (Hall Scragg,2012). Critics of this theory say it feeds into the negative stereotypes of ageing, making it seem acceptable and normal for older people to disengage and therefore preventing them from creating circumstances that increase engagement and quality of life (Bengtson DeLiema, 2016). One of its strengths however, is that the development of this theory opened the door to further research, resulting in a clearer view of natural ageing (Bengtson DeLiema,2016). Havighursts (1963) activity theory and Atchleys (1989) continuity theory were both in response to disengagement theory (Carstensen,199 1). Another such modification to disengagement theory was by Streib Schneider (1971), who concluded that disengagement from one sphere of life, does not mean withdrawal from all spheres (Streib Schneider,1971), this made available the idea of new social roles for the aged. As a social worker, applying Streibs version of disengagement activity within disengagement to the case of Hank, Streib emphasis the need for a new role for the retrenched, involving activities that are not mere busy work but should satisfy the social-psychological needs of Hank and be recognised as valid and socially useful pursuits (Streib Schneider,1971). Encouraging Hanks involvement in activities such as the mens shed or volunteer organisations such as meals on wheels or hands on learning (teaching disengaged youths practical skills). These activities differentiate themselves from hobby work because they not only benefit Hank but are also of community benefit. Keeping active in such a way may also lead to renewed encouragement towards employment in other areas. Another challenge to the disengagement perspective is the exchange theory of ageing. This theory draws upon previous works on the social-psychological theory of exchange, and addresses how a decrease in a persons power and status is associated with ageing (Dowd,1975). Dowd presents ageing and social interaction in the image of a cost-benefit model. Dowd reasons that as we age our conduct becomes a burden to ongoing interactions with others. It takes a greater effort to maintain a level of competent contribution, this decreasing competence means those we interact with are getting less benefit in the return they give. The greater the older person is dependent on others, the more they become a burden. Therefore, an older person begins to withdrawal from social life in order to balance the equation of exchange (Lynott Lynott,1996, Bengtson et.al,1997). For Hank, the loss of his job has given him a perceived loss of identity, status and power, not only amongst his social circle as Hanks close friends are still employed, but also within his family. Hank took pride in being the financial support giver for the family but this has been taken away from him, and his wife Jane has had to step up into full-time work. Hank feels he has nothing to offer the workforce or his social circles and therefore has begun to disengage. He is no longer applying for jobs and has withdrawn from contact with people outside his immediate family. The strength of this theory is its power of explanation into the relationship between the costs and rewards within a relationship. It helps us to understand that when we give something to a relationship whether that be employment, friendship, marriage, that to maintain that relationship, we expect something in return (Bengtson et.al,1997). For Hank, this explains his feeling of discouragement, he wants to give but does not feel he has anything to contribute to a work relationship due to his lack of education and decline in physical health this i s also affecting his social network, and family relationship, as he is no longer contributing to the finances. Although this theory is good at explaining relationships on a cost/benefit model, it does not take into account the complexity of relationships and a persons ability to compromise and adapt. It places people in a selfish light willing to give up a relationship if the costs outweigh the benefits. It also doesnt take into account cultural aspects of a relationship and reciprocity of past care or the increased function of older persons assisting the young such as childcare of a grandchild (Poole,2013, Carstensen,1991). Where disengagement theory sees the withdrawal and social isolation of older people as a societal need and of mutual consent, the exchange theory of ageing shows an unequal exchange between older persons and others in society. Disengagement theorists would say the reduction in interaction is fuelled by emotional distancing, whereas exchange looks at the change in dynamics of relationships and interaction (Carstensen,1991). And whilst both theories offer an explanation as to why we decrease in social interaction as we age, both are criticised for their lack of complete empirical data (Carstensen,1991). For Hank, I can see both as factors for his discouragement and disengagement the change in dynamics due to long term job loss has become a trigger for emotional distancing. For a social worker it is important to draw on the knowledge these and other theories gives us in offering a wider understanding of Hanks circumstance, to assist him in moving forward. Challenges and Social Worker Response At 62, Hank hasnt reached what most of us would consider older age, but he is now facing a serious transition point due to the current circumstance of his job loss. Hank is looking ahead at the challenges all Australians face when growing old. Australian society has made significant achievements in the promotion of health and wellbeing of its older citizens. However, our older population still faces many challenges. Some of which include biological and psychological changes general and mental health including dementia care. Other challenges include ageism and social isolation. Ageism can be seen here in discriminatory practices in employment or retrenchment strategies that see people like Hank lose their job or have difficulty in finding new employment (Thompson,2016). Throughout the last century, the Australian government has developed policies, such as those mentioned in previous sections, to enable people to be more self-sufficient in their older years. However, the government fo cus is often on the cost burden of aged care without consideration of protecting human rights, recognising contributions of older Australians and ensuring the needs of social inclusion (Ife,2012, Poole,2013). Although older people may be seen as a burden to society, they actually make substantial contribution. They volunteer an average 4.4hr more a week than working people and are often a valuable source of child-care to their grand-children (Poole,2013). Social work can play an important role in responding to the needs of a client such as Hank by taking a holistic approach to practice. Holistic care means focusing on the whole person, physical, emotional and spiritual, as it relates to their environment. It is taking into consideration a wide range of factors that affect the client (Tanner Harris, 2008). A social worker will consider all aspects involved in their care including, ecological and systems perspectives, legal and ethical matters, plus the biopsychosocial aspects (AASW,2013). The quality of a persons life can be enriched by adding opportunities to thrive under any circumstances. So assisting Hank maintain his independence and to participate to the fullness of his being, is important to his wellbeing and speaks of Hank in terms of his right to dignity and self-fulfilment. As we have seen above,a social worker uses knowledge, theoretical frameworks and models to identifying the best way to approach and discover the clients, strengths, weakness, and opportunities for participation and well-being. It is also important in the case of Hank, to be up-to-date with the latest policies and procedures provided by both State and Federal government. A person-centred approach considers the client as the expert in their own life and able to decide on their own lives path (Tanner Harris, 2008). Thus, encouraging clients within the system to take control and be empowered within their own lives. The effectiveness of this in Hanks case, is it supports the need for collaboration and keeps at the forefront the client and his family, as people with needs and desires, who should always be in control of their own decisions. Social workers also focus on social justice, delving into the inequalities and oppression within society based on such factors as age, gender, class, poverty, race, culture and sexual orientation to become an advocate of change (AASW,2013, Thompson,2016). They can help develop those opportunities through change in policy, social planning and community involvement (Hughes Heycox,2010). This Contributes not only to the individual client, but to the community as a whole. Conclusion With Australias growing older population and the fast-paced technological world of modernisation and globalisation, there are many real cases like Hanks happening every day. It is hoped that any new federal government reforms will assist in helping older Australians maintain active and productive lives, not only for the sake of individual wellbeing but also for the betterment of society. Social workers can have a role to play in policy reform by being strong voice in advocacy for change. Social Workers also have an arsenal of tools to draw upon, to help work collaboratively with the client to assist them in making decisions that have a positive influence on their lives and the lives of those around them. 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